Telomere erosion in human pluripotent stem cells leads to ATR-mediated mitotic catastrophe.

It is well established that short telomeres activate an ATM-driven DNA damage response that leads to senescence in terminally differentiated cells. However, technical limitations have hampered our understanding of how telomere shortening is signaled in human stem cells. Here, we show that telomere attrition induces ssDNA accumulation (G-strand) at telomeres in human pluripotent stem cells (hPSCs), but not in their differentiated progeny. This led to a unique role for ATR in the response of hPSCs to telomere shortening that culminated in an extended S/G2 cell cycle phase and a longer period of mitosis, which was associated with aneuploidy and mitotic catastrophe. Loss of p53 increased resistance to death, at the expense of increased mitotic abnormalities in hPSCs. Taken together, our data reveal an unexpected dominant role of ATR in hPSCs, combined with unique cell cycle abnormalities and, ultimately, consequences distinct from those observed in their isogenic differentiated counterparts.

Telomere Dysfunction Activates p53 and Represses HNF4α Expression Leading to Impaired Human Hepatocyte Development and Function.

BACKGROUND AND AIMS: Telomere attrition is a major risk factor for end-stage liver disease. Due to a lack of adequate models and intrinsic difficulties in studying telomerase in physiologically relevant cells, the molecular mechanisms responsible for liver disease in patients with telomere syndromes remain elusive. To circumvent that, we used genome editing to generate isogenic human embryonic stem cells (hESCs) harboring clinically relevant mutations in telomerase and subjected them to an in vitro, stage-specific hepatocyte differentiation protocol that resembles hepatocyte development in vivo. APPROACH AND RESULTS: Using this platform, we observed that while telomerase is highly expressed in hESCs, it is quickly silenced, specifically due to telomerase reverse transcriptase component (TERT) down-regulation, immediately after endoderm differentiation and completely absent in in vitro-derived hepatocytes, similar to what is observed in human primary hepatocytes. While endoderm derivation is not impacted by telomere shortening, progressive telomere dysfunction impaired hepatic endoderm formation. Consequently, hepatocyte derivation, as measured by expression of specific hepatic markers as well by albumin expression and secretion, is severely compromised in telomerase mutant cells with short telomeres. Interestingly, this phenotype was not caused by cell death induction or senescence. Rather, telomere shortening prevents the up-regulation and activation of human hepatocyte nuclear factor 4 alpha (HNF4α) in a p53-dependent manner. Both reactivation of telomerase and silencing of p53 rescued hepatocyte formation in telomerase mutants. Likewise, the conditional expression (doxycycline-controlled) of HNF4α, even in cells that retained short telomeres, accrued DNA damage, and exhibited p53 stabilization, successfully restored hepatocyte formation from hESCS. CONCLUSIONS: Our data show that telomere dysfunction acts as a major regulator of HNF4α during hepatocyte development, pointing to a target in the treatment of liver disease in telomere-syndrome patients.

Cognitive function is preserved in aged mice following long-term ß-hydroxy ß-methylbutyrate supplementation.

ß-hydroxy ß-methylbutyrate (HMB) is a nutritional supplement purported to enhance skeletal muscle mass and strength, as well as cognitive function in older adults. The purpose of this study was to determine the potential for long-term HMB supplementation to preserve muscle function and cognition in aged mice, as well as provide evidence of a link between vessel-associated pericyte function and outcomes. Four- (Adult/Ad) and 17 month-old (Aged/Ag) C57BL/6J mice consumed chow containing 600 mg/kg BW/day of either Ca-HMB (Ad, n=16; Ag, n=17) or Ca-Lactate (Ad, n=16; Ag, n=17) for 6 months. HMB did not prevent age-related reductions in muscle mass, strength and coordination (Age main effect, P<0.05). The rate of muscle protein synthesis decreased within the mitochondrial fraction (age main effect, P<0.05), and this decline was not prevented with HMB. Despite no change in muscle mass or function, an age-dependent reduction in active avoidance learning was attenuated with HMB (Age and HMB main effects, P<0.05). Age detrimentally impacted muscle-resident pericyte gene expression with no recovery observed with HMB, whereas no changes in brain-resident pericyte quantity or function were observed with age or HMB. The findings from this study suggest that prolonged HMB supplementation starting in adulthood may preserve cognition with age.

The impact of skeletal muscle contraction on CD146+Lin- pericytes.

Unaccustomed resistance exercise can initiate skeletal muscle remodeling and adaptive mechanisms that can confer protection from damage and enhanced strength with subsequent stimulation. The myofiber may provide the primary origin for adaptation, yet multiple mononuclear cell types within the surrounding connective tissue may also contribute. The purpose of this study was to evaluate the acute response of muscle-resident interstitial cells to contraction initiated by electrical stimulation (e-stim) and subsequently determine the contribution of pericytes to remodeling as a result of training. Mice were subjected to bilateral e-stim or sham treatment. Following a single session of e-stim, NG2+CD45-CD31- (NG2+Lin-) pericyte, CD146+Lin- pericyte, and PDGFRα+ fibroadipogenic progenitor cell quantity and function were evaluated via multiplex flow cytometry and targeted quantitative PCR. Relative quantity was not significantly altered 24 h postcontraction, yet unique gene signatures were observed for each cell population at 3 h postcontraction. CD146+Lin- pericytes appeared to be most responsive to contraction, and upregulation of genes related to immunomodulation and extracellular matrix remodeling was observed via RNA sequencing. Intramuscular injection of CD146+Lin- pericytes did not significantly increase myofiber size yet enhanced ECM remodeling and angiogenesis in response to repeated bouts of e-stim for 4 wk. The results from this study provide the first evidence that CD146+Lin- pericytes are responsive to skeletal muscle contraction and may contribute to the beneficial outcomes associated with exercise.

Pericyte transplantation improves skeletal muscle recovery following hindlimb immobilization.

Conditions of extended bed rest and limb immobilization can initiate rapid and significant loss of skeletal muscle mass and function. Physical rehabilitation is standard practice following a period of disuse, yet mobility may be severely compromised, and recovery is commonly delayed or incomplete in special populations. Thus, a novel approach toward recovery of muscle mass is highly desired. Pericytes [neuron-glial antigen 2 (NG2)+CD31-CD45- (Lineage- [Lin-]) and CD146+Lin-] demonstrate capacity to facilitate muscle repair, yet the ability to enhance myofiber growth following disuse is unknown. In the current study, 3-4-mo-old mice were unilaterally immobilized for 14 d (IM) or immobilized for 14 d followed by 14 d of remobilization (RE). Flow cytometry and targeted gene expression analyses were completed to assess pericyte quantity and function following IM and RE. In addition, a transplantation study was conducted to assess the impact of pericytes on recovery. Results from targeted analyses suggest minimal impact of disuse on pericyte gene expression, yet NG2+Lin- pericyte quantity is reduced following IM (P < 0.05). Remarkably, pericyte transplantation recovered losses in myofiber cross-sectional area and the capillary-to-fiber ratio following RE, whereas deficits remained with vehicle alone (P = 0.01). These findings provide the first evidence that pericytes effectively rehabilitate skeletal muscle mass following disuse atrophy.-Munroe, M., Dvoretskiy, S., Lopez, A., Leong, J., Dyle, M. C., Kong, H., Adams, C. M., Boppart, M. D. Pericyte transplantation improves skeletal muscle recovery following hindlimb immobilization.

α7ß1 Integrin regulation of gene transcription in skeletal muscle following an acute bout of eccentric exercise.

The α7ß1 integrin is concentrated at the costameres of skeletal muscle and provides a critical link between the actin cytoskeleton and laminin in the basement membrane. We previously demonstrated that expression of the α7BX2 integrin subunit (MCK:α7BX2) preserves muscle integrity and enhances myofiber cross-sectional area following eccentric exercise. The purpose of this study was to utilize gene expression profiling to reveal potential mechanisms by which the α7BX2-integrin contributes to improvements in muscle growth after exercise. A microarray analysis was performed using RNA extracted from skeletal muscle of wild-type or transgenic mice under sedentary conditions and 3 h following an acute bout of downhill running. Genes with false discovery rate probability values below the cutoff of P < 0.05 (n = 73) were found to be regulated by either exercise or transgene expression. KEGG pathway analysis detected upregulation of genes involved in endoplasmic reticulum protein processing with integrin overexpression. Targeted analyses verified increased transcription of Rpl13a, Nosip, Ang, Scl7a5, Gys1, Ndrg2, Hspa5, and Hsp40 as a result of integrin overexpression alone or in combination with exercise (P < 0.05). A significant increase in HSPA5 protein and a decrease in CAAT-enhancer-binding protein homologous protein (CHOP) were detected in transgenic muscle (P < 0.05). In vitro knockdown experiments verified integrin-mediated regulation of Scl7a5 The results from this study suggest that the α7ß1 integrin initiates transcription of genes that allow for protection from stress, including activation of a beneficial unfolded protein response and modulation of protein synthesis, both which may contribute to positive adaptations in skeletal muscle as a result of engagement in eccentric exercise.

Impact of ß-hydroxy ß-methylbutyrate (HMB) on age-related functional deficits in mice.

ß-Hydroxy ß-methylbutyrate (HMB) is a metabolite of the essential amino acid leucine. Recent studies demonstrate a decline in plasma HMB concentrations in humans across the lifespan, and HMB supplementation may be able to preserve muscle mass and strength in older adults. However, the impact of HMB supplementation on hippocampal neurogenesis and cognition remains largely unexplored. The purpose of this study was to simultaneously evaluate the impact of HMB on muscle strength, neurogenesis and cognition in young and aged mice. In addition, we evaluated the influence of HMB on muscle-resident mesenchymal stem/stromal cell (Sca-1+CD45-; mMSC) function to address these cells potential to regulate physiological outcomes. Three month-old (n=20) and 24 month-old (n=18) female C57BL/6 mice were provided with either Ca-HMB or Ca-Lactate in a sucrose solution twice per day for 5.5weeks at a dose of 450mg/kg body weight. Significant decreases in relative peak and mean force, balance, and neurogenesis were observed in aged mice compared to young (age main effects, p≤0.05). Short-term HMB supplementation did not alter activity, balance, neurogenesis, or cognitive function in young or aged mice, yet HMB preserved relative peak force in aged mice. mMSC gene expression was significantly reduced with age, but HMB supplementation was able to recover expression of select growth factors known to stimulate muscle repair (HGF, LIF). Overall, our findings demonstrate that while short-term HMB supplementation does not appear to affect neurogenesis or cognitive function in young or aged mice, HMB may maintain muscle strength in aged mice in a manner dependent on mMSC function.

The Impact of Exercise on Statin-Associated Skeletal Muscle Myopathy.

HMG-CoA reductase inhibitors (statins) are the most effective pharmacological means of reducing cardiovascular disease risk. The most common side effect of statin use is skeletal muscle myopathy, which may be exacerbated by exercise. Hypercholesterolemia and training status are factors that are rarely considered in the progression of myopathy. The purpose of this study was to determine the extent to which acute and chronic exercise can influence statin-induced myopathy in hypercholesterolemic (ApoE-/-) mice. Mice either received daily injections of saline or simvastatin (20 mg/kg) while: 1) remaining sedentary (Sed), 2) engaging in daily exercise for two weeks (novel, Nov), or 3) engaging in daily exercise for two weeks after a brief period of training (accustomed, Acct) (2x3 design, n = 60). Cholesterol, activity, strength, and indices of myofiber damage and atrophy were assessed. Running wheel activity declined in both exercise groups receiving statins (statin x time interaction, p<0.05). Cholesterol, grip strength, and maximal isometric force were significantly lower in all groups following statin treatment (statin main effect, p<0.05). Mitochondrial content and myofiber size were increased and 4-HNE was decreased by exercise (statin x exercise interaction, p<0.05), and these beneficial effects were abrogated by statin treatment. Exercise (Acct and Nov) increased atrogin-1 mRNA in combination with statin treatment, yet enhanced fiber damage or atrophy was not observed. The results from this study suggest that exercise (Nov, Acct) does not exacerbate statin-induced myopathy in ApoE-/- mice, yet statin treatment reduces activity in a manner that prevents muscle from mounting a beneficial adaptive response to training.

Diet-induced obesity regulates adipose-resident stromal cell quantity and extracellular matrix gene expression.

Adipose tissue expansion during periods of excess nutrient intake requires significant turnover of the extracellular matrix (ECM) to allow for maximal lipid filling. Recent data suggest that stromal cells may be a primary contributor to ECM modifications in visceral adipose. The purpose of this study was to investigate the capacity for high fat diet (HFD)-induced obesity to alter adipose-derived stromal cell (ADSC) relative quantity and ECM gene expression, and determine the extent to which exercise training can mitigate such changes. Male C57BL/6J mice were placed on control or HFD for 8weeks prior to and following initiation of a 16week treadmill exercise program. ADSCs (Sca-1(+)CD45(-)) were isolated from epididymal adipose tissue and mRNA was evaluated using high throughput qPCR. Stromal cells were also obtained from skeletal muscle (MDSC). HFD decreased the quantity of ADSCs and markedly altered gene expression related to ECM remodeling (Col1α1, MMP2, MMP9, Timp1). Exercise did not reverse these changes. MDSCs were minimally altered by HFD or exercise. Overall, the data from this study suggest that ADSCs decrease in quantity and contribute to adipose ECM remodeling in response to obesity, and exercise training does not significantly impact these outcomes.