A dual role for integrin‐linked kinase and β1‐integrin in modulating cardiac aging

Cardiac performance decreases with age, which is a major risk factor for cardiovascular disease and mortality in the aging human population, but the molecular mechanisms underlying cardiac aging are still poorly understood. Investigating the role of integrin‐linked kinase (ilk) and β1‐integrin (myospheroid, mys) in Drosophila, which colocalize near cardiomyocyte contacts and Z‐bands, we find that reduced ilk or mys function prevents the typical changes of cardiac aging seen in wildtype, such as arrhythmias. In particular, the characteristic increase in cardiac arrhythmias with age is prevented in ilk and mys heterozygous flies with nearly identical genetic background, and they live longer, in line with previous findings in Caenorhabditis elegans for ilk and in Drosophila for mys. Consistent with these findings, we observed elevated β1‐integrin protein levels in old compared with young wild‐type flies, and cardiac‐specific overexpression of mys in young flies causes aging‐like heart dysfunction. Moreover, moderate cardiac‐specific knockdown of integrin‐linked kinase (ILK)/integrin pathway‐associated genes also prevented the decline in cardiac performance with age. In contrast, strong cardiac knockdown of ilk or ILK‐associated genes can severely compromise cardiac integrity, including cardiomyocyte adhesion and overall heart function. These data suggest that ilk/mys function is necessary for establishing and maintaining normal heart structure and function, and appropriate fine‐tuning of this pathway can retard the age‐dependent decline in cardiac performance and extend lifespan. Thus, ILK/integrin‐associated signaling emerges as an important and conserved genetic mechanism in longevity, and as a new means to improve age‐dependent cardiac performance, in addition to its vital role in maintaining cardiac integrity.     

Proteomic profiling of cardiomyopathic tissue from the aged mdx model of Duchenne muscular dystrophy reveals a drastic decrease in laminin,nidogen and annexin

The majority of patients afflicted with Duchenne muscular dystrophy develop cardiomyopathic complications, warranting large‐scale proteomic studies of global cardiac changes for the identification of new protein markers of dystrophinopathy. The aged heart from the X‐linked dystrophic mdx mouse has been shown to exhibit distinct pathological aspects of cardiomyopathy. In order to establish age‐related alterations in the proteome of dystrophin‐deficient hearts, cardiomyopathic tissue from young versus aged mdx mice was examined by label‐free LC‐MS/MS. Significant age‐dependent alterations were established for 67 proteins, of which 28 proteins were shown to exhibit a lower abundance and 39 proteins were found to be increased in their expression levels. Drastic changes were demonstrated for 17 proteins, including increases in Ig chains and transferrin, and drastic decreases in laminin, nidogen and annexin. An immunblotting survey of young and old wild‐type versus mdx hearts confirmed these proteomic findings and illustrated the effects of natural aging versus dystrophin deficiency. These proteome‐wide alterations suggest a disintegration of the basal lamina structure and cytoskeletal network in dystrophin‐deficient cardiac fibres, increased levels of antibodies in a potential autoimmune reaction of the degenerating heart, compensatory binding of excess iron and a general perturbation of metabolic pathways in dystrophy‐associated cardiomyopathy.     

Deletion of Siah-interacting protein gene in <Emphasis Type="Italic">Drosophila</Emphasis> causes cardiomyopathy

Drosophila is a useful model organism in which the genetics of human diseases, including recent advances in identification of the genetics of heart development and disease in the fly, can be studied. To identify novel genes that cause cardiomyopathy, we performed a deficiency screen in adult Drosophila. Using optical coherence tomography to phenotype cardiac function in awake adult Drosophila, we identified Df(1)Exel6240 as having cardiomyopathy. Using a number of strategies including customized smaller deletions, screening of mutant alleles, and transgenic rescue, we identified CG3226 as the causative gene for this deficiency. CG3226 is an uncharacterized gene in Drosophila possessing homology to the mammalian Siah-interacting protein (SIP) gene. Mammalian SIP functions as an adaptor protein involved in one of the β-catenin degradation complexes. To investigate the effects of altering β-catenin/Armadillo signaling in the adult fly, we measured heart function in flies expressing either constitutively active Armadillo or transgenic constructs that block Armadillo signaling, specifically in the heart. While, increasing Armadillo signaling in the heart did not have an effect on adult heart function, decreasing Armadillo signaling in the fly heart caused the significant reduction in heart chamber size. In summary, we show that deletion of CG3226, which has homology to mammalian SIP, causes cardiomyopathy in adult Drosophila. Alterations in Armadillo signaling during development lead to important changes in the size and function of the adult heart.     

Forkhead box O3 protects the heart against paraquat‐induced aging‐associated phenotypes by upregulating the expression of antioxidant enzymes

Paraquat (PQ) promotes cell senescence in brain tissue, which contributes to Parkinson's disease. Furthermore, PQ induces heart failure and oxidative damage, but it remains unknown whether and how PQ induces cardiac aging. Here, we demonstrate that PQ induces phenotypes associated with senescence of cardiomyocyte cell lines and results in cardiac aging‐associated phenotypes including cardiac remodeling and dysfunction in vivo. Moreover, PQ inhibits the activation of Forkhead box O3 (FoxO3), an important longevity factor, both in vitro and in vivo. We found that PQ‐induced senescence phenotypes, including proliferation inhibition, apoptosis, senescence‐associated β‐galactosidase activity, and p16^INK4a expression, were significantly enhanced by FoxO3 deficiency in cardiomyocytes. Notably, PQ‐induced cardiac remolding, apoptosis, oxidative damage, and p16^INK4a expression in hearts were exacerbated by FoxO3 deficiency. In addition, both in vitro deficiency and in vivo deficiency of FoxO3 greatly suppressed the activation of antioxidant enzymes including catalase (CAT) and superoxide dismutase 2 (SOD2) in the presence of PQ, which was accompanied by attenuation in cardiac function. The direct in vivo binding of FoxO3 to the promoters of the Cat and Sod2 genes in the heart was verified by chromatin immunoprecipitation (ChIP). Functionally, overexpression of Cat or Sod2 alleviated the PQ‐induced senescence phenotypes in FoxO3‐deficient cardiomyocyte cell lines. Overexpression of FoxO3 and CAT in hearts greatly suppressed the PQ‐induced heart injury and phenotypes associated with aging. Collectively, these results suggest that FoxO3 protects the heart against an aging‐associated decline in cardiac function in mice exposed to PQ, at least in part by upregulating the expression of antioxidant enzymes and suppressing oxidative stress.     

Age‐associated de‐repression of retrotransposons in the Drosophila fat body,its potential cause and consequence

Eukaryotic genomes contain transposable elements (TE) that can move into new locations upon activation. Since uncontrolled transposition of TEs, including the retrotransposons and DNA transposons, can lead to DNA breaks and genomic instability, multiple mechanisms, including heterochromatin‐mediated repression, have evolved to repress TE activation. Studies in model organisms have shown that TEs become activated upon aging as a result of age‐associated deregulation of heterochromatin. Considering that different organisms or cell types may undergo distinct heterochromatin changes upon aging, it is important to identify pathways that lead to TE activation in specific tissues and cell types. Through deep sequencing of isolated RNAs, we report an increased expression of many retrotransposons in the old Drosophila fat body, an organ equivalent to the mammalian liver and adipose tissue. This de‐repression correlates with an increased number of DNA damage foci and decreased level of Drosophila lamin‐B in the old fat body cells. Depletion of the Drosophila lamin‐B in the young or larval fat body results in a reduction of heterochromatin and a corresponding increase in retrotransposon expression and DNA damage. Further manipulations of lamin‐B and retrotransposon expression suggest a role of the nuclear lamina in maintaining the genome integrity of the Drosophila fat body by repressing retrotransposons.     

MicroRNA-18 and microRNA-19 regulate CTGF and TSP-1 expression in age-related heart failure

To understand the process of cardiac aging, it is of crucial importance to gain insight into the age‐related changes in gene expression in the senescent failing heart. Age‐related cardiac remodeling is known to be accompanied by changes in extracellular matrix (ECM) gene and protein levels. Small noncoding microRNAs regulate gene expression in cardiac development and disease and have been implicated in the aging process and in the regulation of ECM proteins. However, their role in age‐related cardiac remodeling and heart failure is unknown. In this study, we investigated the aging‐associated microRNA cluster 17–92, which targets the ECM proteins connective tissue growth factor (CTGF) and thrombospondin‐1 (TSP‐1). We employed aged mice with a failure‐resistant (C57Bl6) and failure‐prone (C57Bl6 × 129Sv) genetic background and extrapolated our findings to human age‐associated heart failure. In aging‐associated heart failure, we linked an aging‐induced increase in the ECM proteins CTGF and TSP‐1 to a decreased expression of their targeting microRNAs 18a, 19a, and 19b, all members of the miR‐17–92 cluster. Failure‐resistant mice showed an opposite expression pattern for both the ECM proteins and the microRNAs. We showed that these expression changes are specific for cardiomyocytes and are absent in cardiac fibroblasts. In cardiomyocytes, modulation of miR‐18/19 changes the levels of ECM proteins CTGF and TSP‐1 and collagens type 1 and 3. Together, our data support a role for cardiomyocyte‐derived miR‐18/19 during cardiac aging, in the fine‐tuning of cardiac ECM protein levels. During aging, decreased miR‐18/19 and increased CTGF and TSP‐1 levels identify the failure‐prone heart.     

The selective autophagy receptor SQSTM1/p62 improves lifespan and proteostasis in an evolutionarily conserved manner

ABSTRACT

The degradation of specific cargos such as ubiquitinated protein aggregates and dysfunctional mitochondria via macroautophagy/autophagy is facilitated by SQSTM1/p62, the first described selective autophagy receptor in metazoans. While the general process of autophagy plays crucial roles during aging, it remains unclear whether and how selective autophagy mediates effects on longevity and health. Two recent studies in the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster observed gene expression changes of the respective SQSTM1 orthologs in response to environmental stressors or age and showed that overexpression of SQSTM1 is sufficient to extend lifespan and improve proteostasis and mitochondrial function in an autophagy-dependent manner in these model organisms. These findings show that increased expression of the selective autophagy receptor SQSTM1 is sufficient to induce aggrephagy in C. elegans, and mitophagy in Drosophila, and demonstrate an evolutionarily conserved role for SQSTM1 in lifespan determination.     

Increasing autophagy and blocking Nrf2 suppress laminopathy‐induced age‐dependent cardiac dysfunction and shortened lifespan

Mutations in the human LMNA gene cause a collection of diseases known as laminopathies. These include myocardial diseases that exhibit age‐dependent penetrance of dysrhythmias and heart failure. The LMNA gene encodes A‐type lamins, intermediate filaments that support nuclear structure and organize the genome. Mechanisms by which mutant lamins cause age‐dependent heart defects are not well understood. To address this issue, we modeled human disease‐causing mutations in the Drosophila melanogaster Lamin C gene and expressed mutant Lamin C exclusively in the heart. This resulted in progressive cardiac dysfunction, loss of adipose tissue homeostasis, and a shortened adult lifespan. Within cardiac cells, mutant Lamin C aggregated in the cytoplasm, the CncC(Nrf2)/Keap1 redox sensing pathway was activated, mitochondria exhibited abnormal morphology, and the autophagy cargo receptor Ref2(P)/p62 was upregulated. Genetic analyses demonstrated that simultaneous over‐expression of the autophagy kinase Atg1 gene and an RNAi against CncC eliminated the cytoplasmic protein aggregates, restored cardiac function, and lengthened lifespan. These data suggest that simultaneously increasing rates of autophagy and blocking the Nrf2/Keap1 pathway are a potential therapeutic strategy for cardiac laminopathies.     

Attenuation of epigenetic regulator SMARCA4 and ERK‐ETS signaling suppresses aging‐related dopaminergic degeneration

How complex interactions of genetic, environmental factors and aging jointly contribute to dopaminergic degeneration in Parkinson's disease (PD) is largely unclear. Here, we applied frequent gene co‐expression analysis on human patient substantia nigra‐specific microarray datasets to identify potential novel disease‐related genes. In vivo Drosophila studies validated two of 32 candidate genes, a chromatin‐remodeling factor SMARCA4 and a biliverdin reductase BLVRA. Inhibition of SMARCA4 was able to prevent aging‐dependent dopaminergic degeneration not only caused by overexpression of BLVRA but also in four most common Drosophila PD models. Furthermore, down‐regulation of SMARCA4 specifically in the dopaminergic neurons prevented shortening of life span caused by α‐synuclein and LRRK2. Mechanistically, aberrant SMARCA4 and BLVRA converged on elevated ERK‐ETS activity, attenuation of which by either genetic or pharmacological manipulation effectively suppressed dopaminergic degeneration in Drosophila in vivo. Down‐regulation of SMARCA4 or drug inhibition of MEK/ERK also mitigated mitochondrial defects in PINK1 (a PD‐associated gene)‐deficient human cells. Our findings underscore the important role of epigenetic regulators and implicate a common signaling axis for therapeutic intervention in normal aging and a broad range of age‐related disorders including PD.