Senescent cells play important roles in both physiological and pathological processes, including cancer and aging. In all cases, however, senescent cells comprise only a small fraction of tissues. Senescent phenotypes have been studied largely in relatively homogeneous populations of cultured cells. In vivo, senescent cells are generally identified by a small number of markers, but whether and how these markers vary among individual cells is unknown. We therefore utilized a combination of single-cell isolation and a nanofluidic PCR platform to determine the contributions of individual cells to the overall gene expression profile of senescent human fibroblast populations. Individual senescent cells were surprisingly heterogeneous in their gene expression signatures. This cell-to-cell variability resulted in a loss of correlation among the expression of several senescence-associated genes. Many genes encoding senescence-associated secretory phenotype (SASP) factors, a major contributor to the effects of senescent cells in vivo, showed marked variability with a subset of highly induced genes accounting for the increases observed at the population level. Inflammatory genes in clustered genomic loci showed a greater correlation with senescence compared to nonclustered loci, suggesting that these genes are coregulated by genomic location. Together, these data offer new insights into how genes are regulated in senescent cells and suggest that single markers are inadequate to identify senescent cells in vivo.

A decline in skeletal muscle mass and function with aging is well recognized, but remains poorly characterized at the molecular level. Here, we report for the first time a genome-wide study of DNA methylation dynamics in skeletal muscle of healthy male individuals during normal human aging. We predominantly observed hypermethylation throughout the genome within the aged group as compared to the young subjects. Differentially methylated CpG (dmCpG) nucleotides tend to arise intragenically and are underrepresented in promoters and are overrepresented in the middle and 3' end of genes. The intragenic methylation changes are overrepresented in genes that guide the formation of the junction of the motor neuron and myofibers. We report a low level of correlation of gene expression from previous studies of aged muscle with our current analysis of DNA methylation status. For those genes that had both changes in methylation and gene expression with age, we observed a reverse correlation, with the exception of intragenic hypermethylated genes that were correlated with an increased gene expression. We suggest that a minimal number of dmCpG sites or select sites are required to be altered in order to correlate with gene expression changes. Finally, we identified 500 dmCpG sites that perform well in discriminating young from old samples. Our findings highlight epigenetic links between aging postmitotic skeletal muscle and DNA methylation.

Developing accurate methods to quantify age-related muscle loss (sarcopenia) could greatly accelerate development of therapies to treat muscle loss in the elderly, as current methods are inaccurate or expensive. The current gold standard method for quantifying sarcopenia is dual-energy X-ray absorptiometry (DXA) but does not measure muscle directly-it is a composite measure quantifying "lean mass" (muscle) excluding fat and bone. In humans, DXA overestimates muscle mass, which has led to erroneous conclusions about the importance of skeletal muscle in human health and disease. In animal models, DXA is a popular method for measuring lean mass. However, instrumentation is expensive and is potentially limited by anesthesia concerns. Recently, the D3 -creatine (D3 Cr) dilution method for quantifying muscle mass was developed in humans and rats. This method is faster, cheaper, and more accurate than DXA. Here, we demonstrate that the D3 Cr method is a specific assay for muscle mass in mice, and we test associations with DXA and body weight. We evaluated the D3 Cr method compared to DXA-determined lean body mass (LBM) in aged mice and reported that DXA consistently overestimates muscle mass with age. Overall, we provide evidence that the D3 Cr dilution method directly measures muscle mass in mice. Combined with its ease of use, accessibility, and non-invasive nature, the method may prove to more quickly advance development of preclinical therapies targeting sarcopenia.

The nematode Caenorhabditis elegans has become one of the most widely used model systems for the study of aging, yet very little is known about how C. elegans age. The development of the worm, from egg to young adult has been completely mapped at the cellular level, but such detailed studies have not been extended throughout the adult lifespan. Numerous single gene mutations, drug treatments and environmental manipulations have been found to extend worm lifespan. To interpret the mechanism of action of such aging interventions, studies to characterize normal worm aging, similar to those used to study worm development are necessary. We have used 4',6'-diamidino-2-phenylindole hydrochloride staining and quantitative polymerase chain reaction to investigate the integrity of nuclei and quantify the nuclear genome copy number of C. elegans with age. We report both systematic loss of nuclei or nuclear DNA, as well as dramatic age-related changes in nuclear genome copy number. These changes are delayed or attenuated in long-lived daf-2 mutants. We propose that these changes are important pathobiological characteristics of aging nematodes.

Antiretroviral drugs have dramatically improved the prognosis of HIV-infected patients, with strikingly reduced morbidity and mortality. However, long-term use can be associated with signs of premature aging. Highly active antiretroviral therapy generally comprises two nucleoside reverse transcriptase inhibitors (NRTIs), with one of three additional antiretroviral drug classes, including protease inhibitors (PIs). One commonality between mitochondrial dysfunction (induced by NRTIs) and defects in lamin A (induced by PIs) is they can cause or accelerate cellular senescence, a state of essentially irreversible growth arrest, and the secretion of many bioactive molecules collectively known as the senescence-associated secretory phenotype (SASP). We hypothesized that senescent cells increase following treatment with certain HIV therapies. We compared the effects of two distinct HIV PIs: ritonavir-boosted atazanavir (ATV/r) and ritonavir-boosted darunavir (DRN/r), used in combination treatments for HIV infection. Upon ATV/r, but not DRN/r, treatment, cells arrested growth, displayed multiple features of senescence, and expressed significantly upregulated levels of many SASP factors. Furthermore, mice receiving sustained ATV/r treatment showed an increase in senescent cells and age-related decline in physiological function. However, removing treatment reversed the features of senescence observed in vivo and cell culture. Given how these features disappeared with drug removal, certain features of senescence may not be prognostic as defined by an irreversible growth arrest. Importantly, for patients that are treated or have been treated with ATV/r, our data suggest that switching to another PI that does not promote premature aging conditions (DRN/r) may improve the associated age-related complications.

Aging causes cardiac dysfunction, often leading to heart failure and death. The molecular basis of age-associated changes in cardiac structure and function is largely unknown. The fruit fly, Drosophila melanogaster, is well-suited to investigate the genetics of cardiac aging. Flies age rapidly over the course of weeks, benefit from many tools to easily manipulate their genome, and their heart has significant genetic and phenotypic similarities to the human heart. Here, we performed a cardiac-specific gene expression study on aging Drosophila and carried out a comparative meta-analysis with published rodent data. Pathway level transcriptome comparisons suggest that age-related, extra-cellular matrix remodeling and alterations in mitochondrial metabolism, protein handling, and contractile functions are conserved between Drosophila and rodent hearts. However, expression of only a few individual genes similarly changed over time between and even within species. We also examined gene expression in single fly hearts and found significant variability as has been reported in rodents. We propose that individuals may arrive at similar cardiac aging phenotypes via dissimilar transcriptional changes, including those in transcription factors and micro-RNAs. Finally, our data suggest the transcription factor Odd-skipped, which is essential for normal heart development, is also a crucial regulator of cardiac aging.