Rearing and Experimental Uses of Daphnia: Controlling Animal Nutrition and Assessing Lifespan and Life-History Traits.

Caloric restriction has been found to extend the lifespan of many organisms including mammals and other vertebrates. With lifespans exceeding months to years, age-related experiments involving fish and mammals can be overtly costly, both in terms of time and funding. The freshwater crustacean, Daphnia, has a relatively short lifespan (∼50 to 100 days), which makes it a cost-effective alternative animal model for longevity and aging studies. Besides age-specific mortality, there are a suite of physiological responses connected to "healthspan" that can be tracked as these animals age including growth, reproduction, and metabolic rates. These responses can be complemented by assessment of molecular and cellular processes connected to aging and health. Lifespan and metabolism of this model organism is responsive to long studied modulators of aging, such as rearing temperature and nutritional manipulation, but also pharmacological agents that target aging, e.g., rapamycin, which adds to its usefulness as a model organism. Here we describe how to culture Daphnia for aging experiments including maintaining laboratory populations of Daphnia mothers, growing algal food, and manipulating nutrition of these animals. In addition, we provide methods for tracking common physiological and longevity responses of Daphnia. This protocol provides researchers planning to use this model organism with methods to establish and maintain Daphnia populations and to standardize their experimental approaches. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culturing algae for Daphnia food Basic Protocol 2: General methods for culturing Daphnia Basic Protocol 3: Standardizing and controlling nutrition for experimental Daphnia Basic Protocol 4: Monitoring Daphnia lifespan Basic Protocol 5: Evaluating Daphnia health: Heart rate and respiration, body mass and growth rates, and reproduction.

Profiling of adenine-derived signaling molecules, cytokinins, in myotubes reveals fluctuations in response to lipopolysaccharide-induced cell stress.

Cytokinins (CTKs) are a diverse collection of evolutionarily conserved adenine-derived signaling molecules classically studied as phytohormones; however, their roles and production have been less studied in mammalian systems. Skeletal muscles are sensitive to cellular cues such as inflammation and in response, alter their secretome to regulate the muscle stem cell and myofiber niche. Using cultured C2C12 muscle cells, we profiled CTK levels to understand (1) whether CTKs are part of the muscle secretome and (2) whether CTKs are responsive to cellular stress. To induce cellular stress, C2C12 myotubes were treated with lipopolysaccharides (LPS) for 24 h and then media and cell fractions were collected for ultra high-performance liquid chromatography tandem mass spectrometry with electrospray ionization (UHPLC-(ESI+)-HRMS/MS) for metabolomics and CTK profiling. Across LPS-treated and control cells, 11 CTKs were detected in the extracellular space while 6 were detected intracellularly. We found that muscle cells are enriched in isopentenyladenine (iP) species (from free base, riboside to nucleotide forms), and that extracellular levels are increased after LPS treatment. Our study establishes that muscle cells express various forms of CTKs, and that CTK levels are responsive to LPS-induced cell stress, suggesting a role for CTKs in intra- and extracellular signaling of mammalian cells.

The importance of biological sex in cardiac cachexia.

Cardiac cachexia is a catabolic muscle-wasting syndrome observed in approximately 1 in 10 patients with heart failure. Increased skeletal muscle atrophy leads to frailty and limits mobility, which impacts quality of life, exacerbates clinical care, and is associated with higher rates of mortality. Heart failure is known to exhibit a wide range of prevalence and severity when examined across individuals of different ages and with comorbidities related to diabetes, renal failure, and pulmonary dysfunction. It is also recognized that men and women exhibit striking differences in the pathophysiology of heart failure, as well as skeletal muscle homeostasis. Given that both skeletal muscle and heart failure physiology are in part sex-dependent, the diagnosis and treatment of cachexia in patients with heart failure may depend on a comprehensive examination of how these organs interact. In this review, we explore the potential for sex-specific differences in cardiac cachexia. We summarize advantages and disadvantages of clinical methods used to measure muscle mass and function and provide alternative measurements that should be considered in preclinical studies. In addition, we summarize sex-dependent effects on muscle wasting in preclinical models of heart failure, disuse, and cancer. Lastly, we discuss the endocrine function of the heart and outline unanswered questions that could directly impact patient care.

Age-related defects in autophagy alter the secretion of paracrine factors from bone marrow mononuclear cells.

Bone marrow mononuclear cell therapy improves cardiac repair after myocardial infarction (MI), in-part through signaling to resident cardiac cells, such as fibroblasts, which regulate scar formation. The efficacy of cell therapy declines with age, as aging of both donor and recipient cells decreases repair responses. Autophagy regulates the microenvironment by both extracellular vesicle (EV)-dependent and independent secretion pathways. We hypothesized that age-related autophagy changes in bone marrow cells (BMCs) alter paracrine signaling, contributing to lower cell therapy efficacy. Here, we demonstrate that young Sca-1+ BMCs exhibited a higher LC3II/LC3I ratio compared to old Sca-1+ BMCs, which was accentuated when BMCs were cultured under hypoxia. To examine the effect on paracrine signaling, old cardiac fibroblasts were cultured with conditioned medium (CM) from young and old Sca-1+ BMCs. Young, but not old CM, enhanced fibroblast proliferation, migration, and differentiation, plus reduced senescence. These beneficial effects were lost when autophagy or EV secretion in BMCs was blocked pharmacologically, or by siRNA knockdown of Atg7. Therefore, both EV-dependent and -independent paracrine signaling from young BMCs is responsible for paracrine stimulation of old cardiac fibroblasts. In vivo, bone marrow chimerism of old mice with young BMCs increased the number of LC3b+ cells in the heart compared to old mice reconstituted with old BMCs. These data suggest that the deterioration of autophagy with aging negatively impacts the paracrine effects of BMCs, and provide mechanistic insight into the age-related decline in cell therapy efficacy that could be targeted to improve the function of old donor cells.

Delineating the relationship between immune system aging and myogenesis in muscle repair.

Recruited immune cells play a critical role in muscle repair, in part by interacting with local stem cell populations to regulate muscle regeneration. How aging affects their communication during myogenesis is unclear. Here, we investigate how aging impacts the cellular function of these two cell types after muscle injury during normal aging or after immune rejuvenation using a young to old (Y-O) or old to old (O-O) bone marrow (BM) transplant model. We found that skeletal muscle from old mice (20 months) exhibited elevated basal inflammation and possessed fewer satellite cells compared with young mice (3 months). After cardiotoxin muscle injury (CTX), old mice exhibited a blunted inflammatory response compared with young mice and enhanced M2 macrophage recruitment and IL-10 expression. Temporal immune and cytokine responses of old mice were partially restored to a young phenotype following reconstitution with young cells (Y-O chimeras). Improved immune responses in Y-O chimeras were associated with greater satellite cell proliferation compared with O-O chimeras. To identify how immune cell aging affects myoblast function, conditioned media (CM) from activated young or old macrophages was applied to cultured C2C12 myoblasts. CM from young macrophages inhibited myogenesis while CM from old macrophages reduced proliferation. These functional differences coincided with age-related differences in macrophage cytokine expression. Together, this study examines the infiltration and proliferation of immune cells and satellite cells after injury in the context of aging and, using BM chimeras, demonstrates that young immune cells retain cell autonomy in an old host to increase satellite cell proliferation.

Down-Regulation of Lnc-CYP7A1-1 Rejuvenates Aged Human Mesenchymal Stem Cells to Improve Their Efficacy for Heart Repair Through SYNE1.

BACKGROUND: Several long non-coding RNAs (lncRNAs) have been associated with cell senescence, termed senescence-associated lncRNAs (SAL-RNAs). However, the mechanisms involved for SAL-RNAs in aging are not fully elucidated. In the present study, we investigated the effects of SAL-RNAs on aged human bone marrow-derived mesenchymal stem cells (hBM-MSCs), and the possible means to counteract such effects to improve the regenerative capacity of aged hBM-MSCs. METHODS: By comparing the lncRNAs expression of hBM-MSCs derived from young and old individuals, lnc-CYP7A1-1 was identified as being significantly increased with age. Using predictive software, the expression of Spectrin Repeat Containing Nuclear Envelope Protein 1 (SYNE1), was found to be decreased with age. Next, through lentiviral constructs, we downregulated the expression of lnc-CYP7A1-1 or SYNE1 in hBM-MSCs separately. Additionally, hBM-MSCs proliferation, survival, migration, and senescence were investigated in vitro. In vivo, lnc-CYP7A1-1 downregulated aged hBM-MSCs were implanted into infarcted mouse hearts after myocardial infarction (MI), and cardiac function was measured. Through lentivirus-mediated downregulation of lnc-CYP7A1-1 in aged hBM-MSCs, we revealed that cell senescence was decreased, whereas cell proliferation, migration, and survival were increased. On the other hand, downregulation of SYNE1, the target gene of lnc-CYP7A1-1, in young hBM-MSCs increased cell senescence, yet decreased cell proliferation, migration, and survival. Downregulation of lnc-CYP7A1-1 in aged hBM-MSCs induced cell rejuvenation, yet this effect was attenuated by repression of SYNE1. In vivo, transplantation of lnc-CYP7A1-1 downregulated old hBM-MSCs improved cardiac function after MI. CONCLUSION: Down-regulation of lnc-CYP7A1-1 rejuvenated aged hBM-MSCs and improved cardiac function when implanted into the infarcted mouse hearts, possibly through its target gene SYNE1.

Considering Cause and Effect of Immune Cell Aging on Cardiac Repair after Myocardial Infarction.

The importance of the immune system for cardiac repair following myocardial infarction is undeniable; however, the complex nature of immune cell behavior has limited the ability to develop effective therapeutics. This limitation highlights the need for a better understanding of the function of each immune cell population during the inflammatory and resolution phases of cardiac repair. The development of reliable therapies is further complicated by aging, which is associated with a decline in cell and organ function and the onset of cardiovascular and immunological diseases. Aging of the immune system has important consequences on heart function as both chronic cardiac inflammation and an impaired immune response to cardiac injury are observed in older individuals. Several studies have suggested that rejuvenating the aged immune system may be a valid therapeutic candidate to prevent or treat heart disease. Here, we review the basic patterns of immune cell behavior after myocardial infarction and discuss the autonomous and nonautonomous manners of hematopoietic stem cell and immune cell aging. Lastly, we identify prospective therapies that may rejuvenate the aged immune system to improve heart function such as anti-inflammatory and senolytic therapies, bone marrow transplant, niche remodeling and regulation of immune cell differentiation.

Rectification of radiotherapy-induced cognitive impairments in aged mice by reconstituted Sca-1+ stem cells from young donors.

BACKGROUND: Radiotherapy is widely used and effective for treating brain tumours, but inevitably impairs cognition as it arrests cellular processes important for learning and memory. This is particularly evident in the aged brain with limited regenerative capacity, where radiation produces irreparable neuronal damage and activation of neighbouring microglia. The latter is responsible for increased neuronal death and contributes to cognitive decline after treatment. To date, there are few effective means to prevent cognitive deficits after radiotherapy. METHODS: Here we implanted hematopoietic stem cells (HSCs) from young or old (2- or 18-month-old, respectively) donor mice expressing green fluorescent protein (GFP) into old recipients and assessed cognitive abilities 3 months post-reconstitution. RESULTS: Regardless of donor age, GFP+ cells homed to the brain of old recipients and expressed the macrophage/microglial marker, Iba1. However, only young cells attenuated deficits in novel object recognition and spatial memory and learning in old mice post-irradiation. Mechanistically, old recipients that received young HSCs, but not old, displayed significantly greater dendritic spine density and long-term potentiation (LTP) in CA1 neurons of the hippocampus. Lastly, we found that GFP+/Iba1+ cells from young and old donors were differentially polarized to an anti- and pro-inflammatory phenotype and produced neuroprotective factors and reactive nitrogen species in vivo, respectively. CONCLUSION: Our results suggest aged peripherally derived microglia-like cells may exacerbate cognitive impairments after radiotherapy, whereas young microglia-like cells are polarized to a reparative phenotype in the irradiated brain, particularly in neural circuits associated with rewards, learning, and memory. These findings present a proof-of-principle for effectively reinstating central cognitive function of irradiated brains with peripheral stem cells from young donor bone marrow.

Novel mediators of aneurysm progression in bicuspid aortic valve disease.

Bicuspid aortic valve (BAV) disease is a congenital abnormality that is associated with ascending aortic aneurysm yet many of the molecular mechanisms remain unknown. To identify novel molecular mechanisms of aneurysm formation we completed microarray analysis of the proximal (severely dilated) and distal (less dilated) regions of the ascending aorta from five patients with BAV. We identified 180 differentially expressed genes, 40 of which were validated by RT-qPCR. Most genes had roles in inflammation and endothelial cell function including cytokines and growth factors, cell surface receptors and the Activator Protein 1 (AP-1) transcription factor family (FOS, FOSB and JUN) which was chosen for further study. AP-1 was differentially expressed within paired BAV aneurysmal samples (n = 8) but not Marfan patients (n = 5). FOS protein was significantly enriched in BAV aortas compared to normal aortas but unexpectedly, ERK1/2 activity, an upstream regulator of FOS was reduced. ERK1/2 activity was restored when BAV smooth muscle cells were cultured in vitro. An mRNA-miRNA network within paired patient samples identified AP-1 as a central hub of miRNA regulation. FOS knockdown in BAV SMCs increased expression of miR-27a, a stretch responsive miRNA. AP-1 and miR-27a were also dysregulated in a mouse model of aortic constriction. In summary, this study identified a central role for AP-1 signaling in BAV aortic dilatation by using paired mRNA-miRNA patient sample. Upstream analysis of AP-1 regulation showed that the ERK1/2 signaling pathway is dysregulated and thus represents a novel chain of mediators of aortic dilatation in BAV which should be considered in future studies.

Emerging roles of extracellular vesicles in cardiac repair and rejuvenation.

Cell therapy has received significant attention as a therapeutic approach to restore cardiac function after myocardial infarction. Accumulating evidence supports that beneficial effects observed with cell therapy are due to paracrine secretion of multiple factors from transplanted cells, which alter the tissue microenvironment and orchestrate cardiac repair processes. Of these paracrine factors, extracellular vesicles (EVs) have emerged as a key effector of cell therapy. EVs regulate cellular function through the transfer of cargo, such as microRNAs and proteins, which act on multiple biological pathways within recipient cells. These discoveries have led to the development of cell-free therapies using EVs to improve cardiac repair after a myocardial infarction. Here, we present an overview of the current use of EVs to enhance cardiac repair after myocardial infarction. We also discuss the emerging use of EVs for rejuvenation-based therapies. Finally, future directions for the use of EVs as therapeutic agents for cardiac regenerative medicine are also discussed.

Dual roles for bone marrow-derived Sca-1 cells in cardiac function.

Recruitment of stem cells from the bone marrow (BM) is an important aspect of cardiac healing that becomes inefficient with age. We investigated the role of young stem cell antigen 1 (Sca-1)-positive BM cells on the aged heart by microarray analysis after BM reconstitution. Sca-1+ and Sca-1- BM cells from young green fluorescent protein (GFP)-positive mice were used to reconstitute the BM of aged mice. Myocardial infarction (MI) was induced 3 mo later. GFP+ cells were more abundant in the BM, blood, and heart of Sca-1+ mice, which corresponded to preserved cardiac function after MI. At baseline, Sca-1+ BM reconstitution increased cardiac expression of serum response factor, vascular endothelial growth factor A, and myogenic genes, but reduced the expression of Il-1ß. After MI, inflammation was identified as a key difference between Sca-1- and Sca-1+ groups, as cytokine expression and cell surface markers associated with inflammatory cells were up-regulated with Sca-1+ reconstitution. Mac-3 and F4/80 staining showed that the postinfarction heart was composed of a mixture of GFP+ (donor) macrophages, GFP- (host) macrophages, and GFP+ cells that did not contribute to the macrophage population. This study demonstrates that Sca-1+ BM cells regulate cardiac healing though an acute inflammatory response and also before injury by stimulating formation of a beneficial cardiac niche.-Tobin, S. W., Li, S.-H., Li, J., Wu, J., Yeganeh, A., Yu, P., Weisel, R. D., Li, R.-K. Dual roles for bone marrow-derived Sca-1 cells in cardiac function.

TGFß-TAZ/SRF signalling regulates vascular smooth muscle cell differentiation.

Vascular smooth muscle cells (VSMCs) do not terminally differentiate; they modulate their phenotype between proliferative and differentiated states, which is a major factor contributing to vascular diseases. TGFß signalling has been implicated in inducing VSMC differentiation, although the exact mechanism remains largely unknown. Our goal was to assess the network of transcription factors involved in the induction of VSMC differentiation, and to determine the role of TAZ in promoting the quiescent VSMC phenotype. TGFß robustly induces VSMC marker genes in 10T1/2 mouse embryonic fibroblast cells and the potent transcriptional regulator TAZ has been shown to retain Smad complexes on DNA. Thus, the role of TAZ in regulation of VSMC differentiation was studied. Using primary aortic VSMCs coupled with siRNA-mediated gene silencing, our studies reveal that TAZ is required for TGFß induction of smooth muscle genes and is also required for the differentiated VSMC phenotype; synergy between TAZ and SRF, and TAZ and Myocardin (MyoC856), in regulating smooth muscle gene activation was observed. These data provide evidence of components of a novel signalling pathway that links TGFß signalling to induction of smooth muscle genes through a mechanism involving regulation of TAZ and SRF proteins. In addition, we report a physical interaction of TAZ and MyoC856. These observations elucidate a novel level of control of VSMC induction which may have implications for vascular diseases and congenital vascular malformations.