Therapeutic potential of alternative splicing in cardiovascular diseases.

RNA splicing is an important RNA processing step required by multiexon protein-coding mRNAs and some noncoding RNAs. Precise RNA splicing is required for maintaining gene and cell function; however, mis-spliced RNA transcripts can lead to loss- or gain-of-function effects in human diseases. Mis-spliced RNAs induced by gene mutations or the dysregulation of splicing regulators may result in frameshifts, nonsense-mediated decay (NMD), or inclusion/exclusion of exons. Genetic animal models have characterised multiple splicing factors required for cardiac development or function. Moreover, sarcomeric and ion channel genes, which are closely associated with cardiovascular function and disease, are hotspots for AS. Here, we summarise splicing factors and their targets that are associated with cardiovascular diseases, introduce some therapies potentially related to pathological AS targets, and raise outstanding questions and future directions in this field.

What we talk about when we talk about spinal cord aging.

Spinal cord-associated disorders are common in the elderly population; however, the mechanisms underlying spinal aging remain elusive. In a recent Nature paper, Sun et al. systemically analyzed aged spines in nonhuman primates and identified a new cluster of CHIT1-positive microglia that drives motor neuron senescence and subsequent spine aging.

[Study of senescence protein p66Shc on myocardial tissue repair in adult mice].

Our previous study has shown that p66Shc plays an important role in the process of myocardial regeneration in newborn mice, and p66Shc deficiency leads to weakened myocardial regeneration in newborn mice. This study aims to explore the role of p66Shc protein in myocardial injury repair after myocardial infarction in adult mice, in order to provide a new target for the treatment of myocardial injury after myocardial infarction. Mouse myocardial infarction models of adult wild-type (WT) and p66Shc knockout (KO) were constructed by anterior descending branch ligation. The survival rate and heart-to-body weight ratio of two models were compared and analyzed. Masson's staining was used to identify scar area of injured myocardial tissue, and myocyte area was determined by wheat germ agglutinin (WGA) staining. TUNEL staining was used to detect the cardiomyocyte apoptosis. The protein expression of brain natriuretic peptide (BNP), a common marker of myocardial hypertrophy, was detected by Western blotting. The results showed that there was no significant difference in survival rate, myocardial scar area, myocyte apoptosis, and heart weight to body weight ratio between the WT and p66ShcKO mice after myocardial infarction surgery. Whereas the protein expression level of BNP in the p66ShcKO mice was significantly down-regulated compared with that in the WT mice. These results suggest that, unlike in neonatal mice, the deletion of p66Shc has no significant effect on myocardial injury repair after myocardial infarction in adult mice.

SIRT6 is an epigenetic repressor of thoracic aortic aneurysms via inhibiting inflammation and senescence.

Thoracic aortic aneurysms (TAAs) develop asymptomatically and are characterized by dilatation of the aorta. This is considered a life-threating vascular disease due to the risk of aortic rupture and without effective treatments. The current understanding of the pathogenesis of TAA is still limited, especially for sporadic TAAs without known genetic mutation. Sirtuin 6 (SIRT6) expression was significantly decreased in the tunica media of sporadic human TAA tissues. Genetic knockout of Sirt6 in mouse vascular smooth muscle cells accelerated TAA formation and rupture, reduced survival, and increased vascular inflammation and senescence after angiotensin II infusion. Transcriptome analysis identified interleukin (IL)-1ß as a pivotal target of SIRT6, and increased IL-1ß levels correlated with vascular inflammation and senescence in human and mouse TAA samples. Chromatin immunoprecipitation revealed that SIRT6 bound to the Il1b promoter to repress expression partly by reducing the H3K9 and H3K56 acetylation. Genetic knockout of Il1b or pharmacological inhibition of IL-1ß signaling with the receptor antagonist anakinra rescued Sirt6 deficiency mediated aggravation of vascular inflammation, senescence, TAA formation and survival in mice. The findings reveal that SIRT6 protects against TAA by epigenetically inhibiting vascular inflammation and senescence, providing insight into potential epigenetic strategies for TAA treatment.

Lysine crotonylation of SERCA2a correlates to cardiac dysfunction and arrhythmia in Sirt1 cardiac-specific knockout mice.

Protein post-translational modifications (PTMs) are important regulators of protein functions and produce proteome complexity. SIRT1 has NAD+-dependent deacylation of acyl-lysine residues. The present study aimed to explore the correlation between lysine crotonylation (Kcr) on cardiac function and rhythm in Sirt1 cardiac-specific knockout (ScKO) mice and related mechanism. Quantitative proteomics and bioinformatics analysis of Kcr were performed in the heart tissue of ScKO mice established with a tamoxifen-inducible Cre-loxP system. The expression and enzyme activity of crotonylated protein were assessed by western blot, co-immunoprecipitation, and cell biology experiment. Echocardiography and electrophysiology were performed to investigate the influence of decrotonylation on cardiac function and rhythm in ScKO mice. The Kcr of SERCA2a was significantly increased on Lys120 (1.973 folds). The activity of SERCA2a decreased due to lower binding energy of crotonylated SERCA2a and ATP. Changes in expression of PPAR-related proteins suggest abnormal energy metabolism in the heart. ScKO mice had cardiac hypertrophy, impaired cardiac function, and abnormal ultrastructure and electrophysiological activities. We conclude that knockout of SIRT1 alters the ultrastructure of cardiac myocytes, induces cardiac hypertrophy and dysfunction, causes arrhythmia, and changes energy metabolism by regulating Kcr of SERCA2a. These findings provide new insight into the role of PTMs in heart diseases.

Sirtuin 2 deficiency aggravates ageing-induced vascular remodelling in humans and mice.

AIMS: The mechanisms underlying ageing-induced vascular remodelling remain unclear. This study investigates the role and underlying mechanisms of the cytoplasmic deacetylase sirtuin 2 (SIRT2) in ageing-induced vascular remodelling. METHODS AND RESULTS: Transcriptome and quantitative real-time PCR data were used to analyse sirtuin expression. Young and old wild-type and Sirt2 knockout mice were used to explore vascular function and pathological remodelling. RNA-seq, histochemical staining, and biochemical assays were used to evaluate the effects of Sirt2 knockout on the vascular transcriptome and pathological remodelling and explore the underlying biochemical mechanisms. Among the sirtuins, SIRT2 had the highest levels in human and mouse aortas. Sirtuin 2 activity was reduced in aged aortas, and loss of SIRT2 accelerated vascular ageing. In old mice, SIRT2 deficiency aggravated ageing-induced arterial stiffness and constriction-relaxation dysfunction, accompanied by aortic remodelling (thickened vascular medial layers, breakage of elastin fibres, collagen deposition, and inflammation). Transcriptome and biochemical analyses revealed that the ageing-controlling protein p66Shc and metabolism of mitochondrial reactive oxygen species (mROS) contributed to SIRT2 function in vascular ageing. Sirtuin 2 repressed p66Shc activation and mROS production by deacetylating p66Shc at lysine 81. Elimination of reactive oxygen species by MnTBAP repressed the SIRT2 deficiency-mediated aggravation of vascular remodelling and dysfunction in angiotensin II-challenged and aged mice. The SIRT2 coexpression module in aortas was reduced with ageing across species and was a significant predictor of age-related aortic diseases in humans. CONCLUSION: The deacetylase SIRT2 is a response to ageing that delays vascular ageing, and the cytoplasm-mitochondria axis (SIRT2-p66Shc-mROS) is important for vascular ageing. Therefore, SIRT2 may serve as a potential therapeutic target for vascular rejuvenation.

Mic60 is essential to maintain mitochondrial integrity and to prevent encephalomyopathy.

Mitochondrial encephalomyopathies (ME) are frequently associated with mutations of mitochondrial DNA, but the pathogenesis of a subset of ME (sME) remains elusive. Here we report that haploinsufficiency of a mitochondrial inner membrane protein, Mic60, causes progressive neurological abnormalities with insulted mitochondrial structure and neuronal loss in mice. In addition, haploinsufficiency of Mic60 reduces mitochondrial membrane potential and cellular ATP production, increases reactive oxygen species, and alters mitochondrial oxidative phosphorylation complexes in neurons in an age-dependent manner. Moreover, haploinsufficiency of Mic60 compromises brain glucose intake and oxygen consumption in mice, resembling human ME syndrome. We further discover that MIC60 protein expression declined significantly in human sME, implying that insufficient MIC60 may contribute for pathogenesis of human ME. Notably, systemic administration of antioxidant N-acetylcysteine largely reverses mitochondrial dysfunctions and metabolic disorders in haplo-insufficient Mic60 mice, also restores neurological abnormal symptom. These results reveal Mic60 is required in the maintenance of mitochondrial integrity and function, and likely a potential therapeutics target for mitochondrial encephalomyopathies.

Single-cell analyses highlight the proinflammatory contribution of C1q-high monocytes to Behçet's disease.

Behçet's disease (BD) is a chronic vasculitis characterized by systemic immune aberrations. However, a comprehensive understanding of immune disturbances in BD and how they contribute to BD pathogenesis is lacking. Here, we performed single-cell and bulk RNA sequencing to profile peripheral blood mononuclear cells (PBMCs) and isolated monocytes from BD patients and healthy donors. We observed prominent expansion and transcriptional changes in monocytes in PBMCs from BD patients. Deciphering the monocyte heterogeneity revealed the accumulation of C1q-high (C1qhi) monocytes in BD. Pseudotime inference indicated that BD monocytes markedly shifted their differentiation toward inflammation-accompanied and C1qhi monocyte-ended trajectory. Further experiments showed that C1qhi monocytes enhanced phagocytosis and proinflammatory cytokine secretion, and multiplatform analyses revealed the significant clinical relevance of this subtype. Mechanistically, C1qhi monocytes were induced by activated interferon-γ (IFN-γ) signaling in BD patients and were decreased by tofacitinib treatment. Our study illustrates the BD immune landscape and the unrecognized contribution of C1qhi monocytes to BD hyperinflammation, showing their potential as therapeutic targets and clinical assessment indexes.

Comprehensive assessment of cellular senescence in the tumor microenvironment.

Cellular senescence (CS), a state of permanent growth arrest, is intertwined with tumorigenesis. Due to the absence of specific markers, characterizing senescence levels and senescence-related phenotypes across cancer types remain unexplored. Here, we defined computational metrics of senescence levels as CS scores to delineate CS landscape across 33 cancer types and 29 normal tissues and explored CS-associated phenotypes by integrating multiplatform data from ~20 000 patients and ~212 000 single-cell profiles. CS scores showed cancer type-specific associations with genomic and immune characteristics and significantly predicted immunotherapy responses and patient prognosis in multiple cancers. Single-cell CS quantification revealed intra-tumor heterogeneity and activated immune microenvironment in senescent prostate cancer. Using machine learning algorithms, we identified three CS genes as potential prognostic predictors in prostate cancer and verified them by immunohistochemical assays in 72 patients. Our study provides a comprehensive framework for evaluating senescence levels and clinical relevance, gaining insights into CS roles in cancer- and senescence-related biomarker discovery.

Restoring nuclear entry of Sirtuin 2 in oligodendrocyte progenitor cells promotes remyelination during ageing.

The age-dependent decline in remyelination potential of the central nervous system during ageing is associated with a declined differentiation capacity of oligodendrocyte progenitor cells (OPCs). The molecular players that can enhance OPC differentiation or rejuvenate OPCs are unclear. Here we show that, in mouse OPCs, nuclear entry of SIRT2 is impaired and NAD+ levels are reduced during ageing. When we supplement ß-nicotinamide mononucleotide (ß-NMN), an NAD+ precursor, nuclear entry of SIRT2 in OPCs, OPC differentiation, and remyelination were rescued in aged animals. We show that the effects on myelination are mediated via the NAD+-SIRT2-H3K18Ac-ID4 axis, and SIRT2 is required for rejuvenating OPCs. Our results show that SIRT2 and NAD+ levels rescue the aged OPC differentiation potential to levels comparable to young age, providing potential targets to enhance remyelination during ageing.

Targeting senescent cells for vascular aging and related diseases.

Cardiovascular diseases are a serious threat to human health, especially in the elderly. Vascular aging makes people more susceptible to cardiovascular diseases due to significant dysfunction or senescence of vascular cells and maladaptation of vascular structure and function; moreover, vascular aging is currently viewed as a modifiable cardiovascular risk factor. To emphasize the relationship between senescent cells and vascular aging, we first summarize the roles of senescent vascular cells (endothelial cells, smooth muscle cells and immune cells) in the vascular aging process and inducers that contribute to cellular senescence. Then, we present potential strategies for directly targeting senescent cells (senotherapy) or preventively targeting senescence inducers (senoprevention) to delay vascular aging and the development of age-related vascular diseases. Finally, based on recent research, we note some important questions that still need to be addressed in the future.

Histone Deacetylase SIRT1, Smooth Muscle Cell Function, and Vascular Diseases.

Vascular smooth muscle cells (VSMCs), located in the media of artery, play key roles in maintaining the normal vascular physiological functions. Abnormality in VSMCs is implicated in vascular diseases (VDs), including atherosclerosis, abdominal aortic aneurysm (AAA), aortic dissection, and hypertension by regulating the process of inflammation, phenotypic switching, and extracellular matrix degradation. Sirtuins (SIRTs), a family of proteins containing seven members (from SIRT1 to SIRT7) in mammals, function as NAD+-dependent histone deacetylases and ADP-ribosyltransferases. In recent decades, great attention has been paid to the cardiovascular protective effects of SIRTs, especially SIRT1, suggesting a new therapeutic target for the treatment of VDs. In this review, we introduce the basic functions of SIRT1 against VSMC senescence, and summarize the contribution of SIRT1 derived from VSMCs in VDs. Finally, the potential new strategies based on SIRT1 activation have also been discussed with an emphasis on SIRT1 activators and calorie restriction to improve the prognosis of VDs.

A research agenda for ageing in China in the 21st century (2nd edition): Focusing on basic and translational research, long-term care, policy and social networks.

One of the key issues facing public healthcare is the global trend of an increasingly ageing society which continues to present policy makers and caregivers with formidable healthcare and socio-economic challenges. Ageing is the primary contributor to a broad spectrum of chronic disorders all associated with a lower quality of life in the elderly. In 2019, the Chinese population constituted 18 % of the world population, with 164.5 million Chinese citizens aged 65 and above (65+), and 26 million aged 80 or above (80+). China has become an ageing society, and as it continues to age it will continue to exacerbate the burden borne by current family and public healthcare systems. Major healthcare challenges involved with caring for the elderly in China include the management of chronic non-communicable diseases (CNCDs), physical frailty, neurodegenerative diseases, cardiovascular diseases, with emerging challenges such as providing sufficient dental care, combating the rising prevalence of sexually transmitted diseases among nursing home communities, providing support for increased incidences of immune diseases, and the growing necessity to provide palliative care for the elderly. At the governmental level, it is necessary to make long-term strategic plans to respond to the pressures of an ageing society, especially to establish a nationwide, affordable, annual health check system to facilitate early diagnosis and provide access to affordable treatments. China has begun work on several activities to address these issues including the recent completion of the of the Ten-year Health-Care Reform project, the implementation of the Healthy China 2030 Action Plan, and the opening of the National Clinical Research Center for Geriatric Disorders. There are also societal challenges, namely the shift from an extended family system in which the younger provide home care for their elderly family members, to the current trend in which young people are increasingly migrating towards major cities for work, increasing reliance on nursing homes to compensate, especially following the outcomes of the 'one child policy' and the 'empty-nest elderly' phenomenon. At the individual level, it is important to provide avenues for people to seek and improve their own knowledge of health and disease, to encourage them to seek medical check-ups to prevent/manage illness, and to find ways to promote modifiable health-related behaviors (social activity, exercise, healthy diets, reasonable diet supplements) to enable healthier, happier, longer, and more productive lives in the elderly. Finally, at the technological or treatment level, there is a focus on modern technologies to counteract the negative effects of ageing. Researchers are striving to produce drugs that can mimic the effects of 'exercising more, eating less', while other anti-ageing molecules from molecular gerontologists could help to improve 'healthspan' in the elderly. Machine learning, 'Big Data', and other novel technologies can also be used to monitor disease patterns at the population level and may be used to inform policy design in the future. Collectively, synergies across disciplines on policies, geriatric care, drug development, personal awareness, the use of big data, machine learning and personalized medicine will transform China into a country that enables the most for its elderly, maximizing and celebrating their longevity in the coming decades. This is the 2nd edition of the review paper (Fang EF et al., Ageing Re. Rev. 2015).

Metabolic regulation of immune cells in proinflammatory microenvironments and diseases during ageing.

The process of ageing includes molecular changes within cells and interactions between cells, eventually resulting in age-related diseases. Although various cells (immune cells, parenchymal cells, fibroblasts and endothelial cells) in tissues secrete proinflammatory signals in age-related diseases, immune cells are the major contributors to inflammation. Many studies have emphasized the role of metabolic dysregulation in parenchymal cells in age-related inflammatory diseases. However, few studies have discussed metabolic modifications in immune cells during ageing. In this review, we introduce the metabolic dysregulation of major nutrients (glucose, lipids, and amino acids) within immune cells during ageing, which leads to dysfunctional NAD + metabolism that increases immune cell senescence and leads to the acquisition of the corresponding senescence-associated secretory phenotype (SASP). We then focus on senescent immune cell interactions with parenchymal cells and the extracellular matrix and their involvement in angiogenesis, which lead to proinflammatory microenvironments in tissues and inflammatory diseases at the systemic level. Elucidating the roles of metabolic modifications in immune cells during ageing will provide new insights into the mechanisms of ageing and therapeutic directions for age-related inflammatory diseases.

Global Lysine Crotonylation Profiling of Mouse Liver.

Lysine crotonylation (Kcr) is a recently discovered post-translational modification that potentially regulates multiple biological processes. With an objective to expand the available crotonylation datasets, LC-MS/MS is performed using mouse liver samples under normal physiological conditions to obtain in vivo crotonylome. A label-free strategy is used and 10 034 Class I (localization probabilities > 0.75) crotonylated sites are identified in 2245 proteins. The KcrE, KcrD, and EKcr motifs are significantly enriched in the crotonylated peptides. The identified crotonylated proteins are mostly enzymes and primarily located in the cytoplasm and nucleus. Functional enrichment analysis based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes shows that the crotonylated proteins are closely related to the purine-containing compound metabolic process, ribose phosphate metabolic process, carbon metabolism pathway, ribosome pathway, and a series of metabolism-associated biological processes. To the best of the authors' knowledge, this research provides the first report on the mouse liver crotonylome. Furthermore, it offers additional evidence that crotonylation exists in non-histone proteins, and is likely involved in various biological processes. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium with the dataset identifiers PXD019145.