The increase in cell death rates in caloric restricted cells of the yeast helicase mutant rrm3 is Sir complex dependent.

Calorie restriction (CR), which is a reduction in calorie intake without malnutrition, usually extends lifespan and improves tissue integrity. This report focuses on the relationship between nuclear genomic instability and dietary-restriction and its effect on cell survival. We demonstrate that the cell survival rates of the genomic instability yeast mutant rrm3 change under metabolic restricted conditions. Rrm3 is a DNA helicase, chromosomal replication slows (and potentially stalls) in its absence with increased rates at over 1400 natural pause sites including sites within ribosomal DNA and tRNA genes. Whereas rrm3 mutant cells have lower cell death rates compared to wild type (WT) in growth medium containing normal glucose levels (i.e., 2%), under CR growth conditions cell death rates increase in the rrm3 mutant to levels, which are higher than WT. The silent-information-regulatory (Sir) protein complex and mitochondrial oxidative stress are required for the increase in cell death rates in the rrm3 mutant when cells are transferred from growth medium containing 2% glucose to CR-medium. The Rad53 checkpoint protein is highly phosphorylated in the rrm3 mutant in response to genomic instability in growth medium containing 2% glucose. Under CR, Rad53 phosphorylation is largely reduced in the rrm3 mutant in a Sir-complex dependent manner. Since CR is an adjuvant treatment during chemotherapy, which may target genomic instability in cancer cells, our studies may gain further insight into how these therapy strategies can be improved.

Deficiency of mitochondrial calcium uniporter abrogates iron overload-induced cardiac dysfunction by reducing ferroptosis.

Iron overload associated cardiac dysfunction remains a significant clinical challenge whose underlying mechanism(s) have yet to be defined. We aim to evaluate the involvement of the mitochondrial Ca2+ uniporter (MCU) in cardiac dysfunction and determine its role in the occurrence of ferroptosis. Iron overload was established in control (MCUfl/fl) and conditional MCU knockout (MCUfl/fl-MCM) mice. LV function was reduced by chronic iron loading in MCUfl/fl mice, but not in MCUfl/fl-MCM mice. The level of mitochondrial iron and reactive oxygen species were increased and mitochondrial membrane potential and spare respiratory capacity (SRC) were reduced in MCUfl/fl cardiomyocytes, but not in MCUfl/fl-MCM cardiomyocytes. After iron loading, lipid oxidation levels were increased in MCUfl/fl, but not in MCUfl/fl-MCM hearts. Ferrostatin-1, a selective ferroptosis inhibitor, reduced lipid peroxidation and maintained LV function in vivo after chronic iron treatment in MCUfl/fl hearts. Isolated cardiomyocytes from MCUfl/fl mice demonstrated ferroptosis after acute iron treatment. Moreover, Ca2+ transient amplitude and cell contractility were both significantly reduced in isolated cardiomyocytes from chronically Fe treated MCUfl/fl hearts. However, ferroptosis was not induced in cardiomyocytes from MCUfl/fl-MCM hearts nor was there a reduction in Ca2+ transient amplitude or cardiomyocyte contractility. We conclude that mitochondrial iron uptake is dependent on MCU, which plays an essential role in causing mitochondrial dysfunction and ferroptosis under iron overload conditions in the heart. Cardiac-specific deficiency of MCU prevents the development of ferroptosis and iron overload-induced cardiac dysfunction.

Bcl-x short-isoform is essential for maintaining homeostasis of multiple tissues.

BCL-2-like protein 1 (BCL2L1) is a key component of cell survival and death mechanisms. Its dysregulation and altered ratio of splicing variants associate with pathologies. However, isoform-specific loss-of-function analysis of BCL2L1 remains unexplored. Here we show the functional impact of genetically inhibiting Bcl-x short-isoform (Bcl-xS) in vivo. Bcl-xS is expressed in most tissues with predominant expression in the spleen and blood cells in mice. Bcl-xS knockout (KO) mice show no overt abnormality until 3 months of age. Thereafter, KO mice develop cardiac hypertrophy with contractile dysfunction and splenomegaly by 6 months. Cardiac fibrosis significantly increases in KO, but the frequency of apoptosis is indistinguishable despite cardiomyopathy. The Akt/mTOR and JNK/cJun signaling are upregulated in male KO heart, and the JNK/cJun is activated with increased Bax expression in KO spleen. These results suggest that Bcl-xS may be dispensable for development but is essential for maintaining the homeostasis of multiple organs.

PERM1 regulates genes involved in fatty acid metabolism in the heart by interacting with PPARα and PGC-1α.

PERM1 (PGC-1/ERR-induced regulator in muscle 1) is a muscle-specific protein induced by PGC-1 and ERRs. Previous studies have shown that PERM1 promotes mitochondrial biogenesis and metabolism in cardiomyocytes in vitro. However, the role of endogenous PERM1 in the heart remains to be investigated with loss-of-function studies in vivo. We report the generation and characterization of systemic Perm1 knockout (KO) mice. The baseline cardiac phenotype of the homozygous Perm1 KO mice appeared normal. However, RNA-sequencing and unbiased pathway analyses showed that homozygous downregulation of PERM1 leads to downregulation of genes involved in fatty acid and carbohydrate metabolism in the heart. Transcription factor binding site analyses suggested that PPARα and PGC-1α are involved in changes in the gene expression profile. Chromatin immunoprecipitation assays showed that PERM1 interacts with the proximal regions of PPAR response elements (PPREs) in endogenous promoters of genes involved in fatty acid oxidation. Co-immunoprecipitation and reporter gene assays showed that PERM1 promoted transcription via the PPRE, partly in a PPARα and PGC-1α dependent manner. These results suggest that endogenous PERM1 is involved in the transcription of genes involved in fatty acid oxidation through physical interaction with PPARα and PGC-1α in the heart in vivo.

Cathepsin D interacts with adenosine A2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling.

Adenosine A2A receptor (A2AR)-dependent signaling in macrophages plays a key role in the regulation of inflammation. However, the processes regulating A2AR targeting to the cell surface and degradation in macrophages are incompletely understood. For example, the C-terminal domain of the A2AR and proteins interacting with it are known to regulate receptor recycling, although it is unclear what role potential A2AR-interacting partners have in macrophages. Here, we aimed to identify A2AR-interacting partners in macrophages that may effect receptor trafficking and activity. To this end, we performed a yeast two-hybrid screen using the C-terminal tail of A2AR as the "bait" and a macrophage expression library as the "prey." We found that the lysosomal protease cathepsin D (CtsD) was a robust hit. The A2AR-CtsD interaction was validated in vitro and in cellular models, including RAW 264.7 and mouse peritoneal macrophage (IPMΦ) cells. We also demonstrated that the A2AR is a substrate of CtsD and that the blockade of CtsD activity increases the density and cell surface targeting of A2AR in macrophages. Conversely, we demonstrate that A2AR activation prompts the maturation and enzymatic activity of CtsD in macrophages. In summary, we conclude that CtsD is a novel A2AR-interacting partner and thus describe molecular and functional interplay that may be crucial for adenosine-mediated macrophage regulation in inflammatory processes.

Ulk1-dependent alternative mitophagy plays a protective role during pressure overload in the heart.

AIMS: Well-controlled mitochondrial homeostasis, including a mitochondria-specific form of autophagy (hereafter referred to as mitophagy), is essential for maintaining cardiac function. The molecular mechanism mediating mitophagy during pressure overload (PO) is poorly understood. We have shown previously that mitophagy in the heart is mediated primarily by Atg5/Atg7-independent mechanisms, including Unc-51-like kinase 1 (Ulk1)-dependent alternative mitophagy, during myocardial ischaemia. Here, we investigated the role of alternative mitophagy in the heart during PO-induced hypertrophy. METHODS AND RESULTS: Mitophagy was observed in the heart in response to transverse aortic constriction (TAC), peaking at 3-5 days. Whereas mitophagy is transiently up-regulated by TAC through an Atg7-dependent mechanism in the heart, peaking at 1 day, it is also activated more strongly and with a delayed time course through an Ulk1-dependent mechanism. TAC induced more severe cardiac dysfunction, hypertrophy, and fibrosis in ulk1 cardiac-specific knock-out (cKO) mice than in wild-type mice. Delayed activation of mitophagy was characterized by the co-localization of Rab9 dots and mitochondria and phosphorylation of Rab9 at Ser179, major features of alternative mitophagy. Furthermore, TAC-induced decreases in the mitochondrial aspect ratio were abolished and the irregularity of mitochondrial cristae was exacerbated, suggesting that mitochondrial quality control mechanisms are impaired in ulk1 cKO mice in response to TAC. TAT-Beclin 1 activates mitophagy even in Ulk1-deficient conditions. TAT-Beclin 1 treatment rescued mitochondrial dysfunction and cardiac dysfunction in ulk1 cKO mice during PO. CONCLUSION: Ulk1-mediated alternative mitophagy is a major mechanism mediating mitophagy in response to PO and plays an important role in mediating mitochondrial quality control mechanisms and protecting the heart against cardiac dysfunction.

AAV-mediated YAP expression in cardiac fibroblasts promotes inflammation and increases fibrosis.

Fibrosis is a hallmark of heart disease independent of etiology and is thought to contribute to impaired cardiac dysfunction and development of heart failure. However, the underlying mechanisms that regulate the differentiation of fibroblasts to myofibroblasts and fibrotic responses remain incompletely defined. As a result, effective treatments to mitigate excessive fibrosis are lacking. We recently demonstrated that the Hippo pathway effector Yes-associated protein (YAP) is an important mediator of myofibroblast differentiation and fibrosis in the infarcted heart. Yet, whether YAP activation in cardiac fibroblasts is sufficient to drive fibrosis, and how fibroblast YAP affects myocardial inflammation, a significant component of adverse cardiac remodeling, are largely unknown. In this study, we leveraged adeno-associated virus (AAV) to target cardiac fibroblasts and demonstrate that chronic YAP expression upregulated indices of fibrosis and inflammation in the absence of additional stress. YAP occupied the Ccl2 gene and promoted Ccl2 expression, which was associated with increased macrophage infiltration, pro-inflammatory cytokine expression, collagen deposition, and cardiac dysfunction in mice with cardiac fibroblast-targeted YAP overexpression. These results are consistent with other recent reports and extend our understanding of YAP function in modulating fibrotic and inflammatory responses in the heart.

Dietary carbohydrates restriction inhibits the development of cardiac hypertrophy and heart failure.

AIMS: A diet with modified components, such as a ketogenic low-carbohydrate (LC) diet, potentially extends longevity and healthspan. However, how an LC diet impacts on cardiac pathology during haemodynamic stress remains elusive. This study evaluated the effects of an LC diet high in either fat (Fat-LC) or protein (Pro-LC) in a mouse model of chronic hypertensive cardiac remodelling. METHODS AND RESULTS: Wild-type mice were subjected to transverse aortic constriction, followed by feeding with the Fat-LC, the Pro-LC, or a high-carbohydrate control diet. After 4 weeks, echocardiographic, haemodynamic, histological, and biochemical analyses were performed. LC diet consumption after pressure overload inhibited the development of pathological hypertrophy and systolic dysfunction compared to the control diet. An anti-hypertrophic serine/threonine kinase, GSK-3ß, was re-activated by both LC diets; however, the Fat-LC, but not the Pro-LC, diet exerted cardioprotection in GSK-3ß cardiac-specific knockout mice. ß-hydroxybutyrate, a major ketone body in mammals, was increased in the hearts of mice fed the Fat-LC, but not the Pro-LC, diet. In cardiomyocytes, ketone body supplementation inhibited phenylephrine-induced hypertrophy, in part by suppressing mTOR signalling. CONCLUSION: Strict carbohydrate restriction suppresses pathological cardiac growth and heart failure after pressure overload through distinct anti-hypertrophic mechanisms elicited by supplemented macronutrients.

Blockade of Fibroblast YAP Attenuates Cardiac Fibrosis and Dysfunction Through MRTF-A Inhibition.

Fibrotic remodeling of the heart in response to injury contributes to heart failure, yet therapies to treat fibrosis remain elusive. Yes-associated protein (YAP) is activated in cardiac fibroblasts by myocardial infarction, and genetic inhibition of fibroblast YAP attenuates myocardial infarction-induced cardiac dysfunction and fibrosis. YAP promotes myofibroblast differentiation and associated extracellular matrix gene expression through engagement of TEA domain transcription factor 1 and subsequent de novo expression of myocardin-related transcription factor A. Thus, fibroblast YAP is a promising therapeutic target to prevent fibrotic remodeling and heart failure.

Acute NelfA knockdown restricts compensatory gene expression and precipitates ventricular dysfunction during cardiac hypertrophy.

Coordinated functional balance of negative and positive transcription complexes maintain and accommodate gene expression in hearts during quiescent and hypertrophic conditions, respectively. Negative elongation factor (Nelf) complex has been implicated in RNA polymerase II (pol II) pausing, a widespread regulatory transcriptional phenomenon observed across the cardiac genome. Here, we examine the role of NelfA aka, Wolf-Hirschhorn syndrome candidate 2 (Whsc2), a critical component of the negative elongation complex in hearts undergoing pressure-overload induced hypertrophy. Alignment of high-resolution genome-wide occupancy data of NelfA, Pol II, TFIIB and H3k9ac from control and hypertrophied hearts reveal that NelfA associates with active gene promoters. High NelfA occupancy is seen at promoters of essential and cardiac-enriched genes, expressed under both quiescent and hypertrophic conditions. Conversely, de novo NelfA recruitment is observed at inducible gene promoters with pressure overload, accompanied by significant increase in expression of these genes with hypertrophy. Interestingly, change in promoter NelfA levels correlates with the transcript output in hypertrophied hearts compared to Sham, suggesting NelfA might be playing a critical role in the regulation of gene transcription during cardiac hypertrophy. In vivo knockdown of NelfA (siNelfA) in hearts subjected to pressure-overload results in early ventricular dilatation and dysfunction, associated with decrease in expression of inducible and cardiac-enriched genes in siNelfA hypertrophied compared to control hypertrophied hearts. In accordance, in vitro knockdown of NelfA in cardiomyocytes showed no change in promoter pol II, however significant decrease in in-gene and downstream pol II occupancy was observed. These data suggest an inhibited pol II progression in transcribing and inducible genes, which reflects as a decrease in transcript abundance of these genes. These results indicate that promoter NelfA occupancy is essential for pol II -dependent transcription. Therefore, we conclude that NelfA is required for active transcription and gene expression during cardiac hypertrophy.

Aging: Fewer Obstacles to DNA Replication?

Many cancers are initiated by loss-of-heterozygosity (LOH) events that lead to the replacement of single, functional tumor suppressor genes by the mutant alleles. The underlying mechanisms, of why LOH rates increase with age, are not well understood. We discuss the possible involvement of difficult-to-replicate (fragile) chromosomal sites in this process.

Absence of Non-histone Protein Complexes at Natural Chromosomal Pause Sites Results in Reduced Replication Pausing in Aging Yeast Cells.

There is substantial evidence that genomic instability increases during aging. Replication pausing (and stalling) at difficult-to-replicate chromosomal sites may induce genomic instability. Interestingly, in aging yeast cells, we observed reduced replication pausing at various natural replication pause sites (RPSs) in ribosomal DNA (rDNA) and non-rDNA locations (e.g., silent replication origins and tRNA genes). The reduced pausing occurs independent of the DNA helicase Rrm3p, which facilitates replication past these non-histone protein-complex-bound RPSs, and is independent of the deacetylase Sir2p. Conditions of caloric restriction (CR), which extend life span, also cause reduced replication pausing at the 5S rDNA and at tRNA genes. In aged and CR cells, the RPSs are less occupied by their specific non-histone protein complexes (e.g., the preinitiation complex TFIIIC), likely because members of these complexes have primarily cytosolic localization. These conditions may lead to reduced replication pausing and may lower replication stress at these sites during aging.

Binding of Multiple Rap1 Proteins Stimulates Chromosome Breakage Induction during DNA Replication.

Telomeres, the ends of linear eukaryotic chromosomes, have a specialized chromatin structure that provides a stable chromosomal terminus. In budding yeast Rap1 protein binds to telomeric TG repeat and negatively regulates telomere length. Here we show that binding of multiple Rap1 proteins stimulates DNA double-stranded break (DSB) induction at both telomeric and non-telomeric regions. Consistent with the role of DSB induction, Rap1 stimulates nearby recombination events in a dosage-dependent manner. Rap1 recruits Rif1 and Rif2 to telomeres, but neither Rif1 nor Rif2 is required for DSB induction. Rap1-mediated DSB induction involves replication fork progression but inactivation of checkpoint kinase Mec1 does not affect DSB induction. Rap1 tethering shortens artificially elongated telomeres in parallel with telomerase inhibition, and this telomere shortening does not require homologous recombination. These results suggest that Rap1 contributes to telomere homeostasis by promoting chromosome breakage.

Reversible mitochondrial DNA accumulation in nuclei of pluripotent stem cells.

According to the endosymbiotic hypothesis, the precursor of mitochondria invaded the precursor of eukaryotic cells, a process that began roughly 2 billion years ago. Since then, the majority of the genetic material translocated from the mitochondria to the nucleus, where now almost all mitochondrial proteins are expressed. Only a tiny amount of DNA remained in the mitochondria, known as mitochondrial DNA (mtDNA). In this study, we report that the transfer of mtDNA fragments to the nucleus of pluripotent stem cells is still ongoing. We show by in situ hybridization and agarose DNA two-dimensional gel technique that induced pluripotent stem (iPS) cells contain high levels of mtDNA in the nucleus. We found that a large proportion of the accumulated mtDNA sequences appear to be extrachromosomal. Accumulation of mtDNA in the nucleus is present not only in the iPS cells, but also in embryonic stem (ES) cells. However upon differentiation, the level of mtDNA in the nuclei of iPS and ES cells is substantially reduced. This reversible accumulation of mtDNA in the nucleus supports the notion that the nuclear copy number of mtDNA sequences may provide a novel mechanism by which chromosomal DNA is dynamically regulated in pluripotent stem cells.

Mst1 inhibits autophagy by promoting the interaction between Beclin1 and Bcl-2.

Here we show that Mst1, a proapoptotic kinase, impairs protein quality control mechanisms in the heart through inhibition of autophagy. Stress-induced activation of Mst1 in cardiomyocytes promoted accumulation of p62 and aggresome formation, accompanied by the disappearance of autophagosomes. Mst1 phosphorylated the Thr108 residue in the BH3 domain of Beclin1, which enhanced the interaction between Beclin1 and Bcl-2 and/or Bcl-xL, stabilized the Beclin1 homodimer, inhibited the phosphatidylinositide 3-kinase activity of the Atg14L-Beclin1-Vps34 complex and suppressed autophagy. Furthermore, Mst1-induced sequestration of Bcl-2 and Bcl-xL by Beclin1 allows Bax to become active, thereby stimulating apoptosis. Mst1 promoted cardiac dysfunction in mice subjected to myocardial infarction by inhibiting autophagy, associated with increased levels of Thr108-phosphorylated Beclin1. Moreover, dilated cardiomyopathy in humans was associated with increased levels of Thr108-phosphorylated Beclin1 and signs of autophagic suppression. These results suggest that Mst1 coordinately regulates autophagy and apoptosis by phosphorylating Beclin1 and consequently modulating a three-way interaction among Bcl-2 proteins, Beclin1 and Bax.

Analysis of DNA structures from eukaryotic cells by two-dimensional native-native DNA agarose gel electrophoresis.

The neutral-neutral two-dimensional agarose gel technique is mainly used to determine the chromosomal positions where DNA replication starts, but it is also applied to visualize replication fork progression and breakage as well as intermediates in DNA recombination. Here we provide a step-by-step protocol to analyze the fairly underrepresented and fragile replication intermediates in yeast chromosomal DNA. The technique can also be adapted to analyze replication intermediates in chromosomal DNA of higher eukaryotic organisms.

Directionality of replication fork movement determined by two-dimensional native-native DNA agarose gel electrophoresis.

The analysis of replication intermediates by the neutral-neutral two-dimensional agarose gel technique allows determining the chromosomal positions where DNA replication initiates, whether replication forks pause or stall at specific sites, or whether two DNA molecules undergo DNA recombination events. This technique does not, however, immediately tell in which direction replication forks migrate through the DNA region under investigation. Here, we describe the procedure to determine the direction of replication fork progression by carrying out a restriction enzyme digest of DNA imbedded in agarose after the completion of the first dimension of a 2D gel.

Accumulation of linear mitochondrial DNA fragments in the nucleus shortens the chronological life span of yeast.

Translocation of mitochondrial DNA (mtDNA) fragments to the nucleus and insertion of those fragments into nuclear DNA has been observed in several organisms ranging from yeast to plants and mammals. Disruption of specific nuclear genes by de novo insertions of mtDNA fragments has even been linked to the initiation of several human diseases. Recently, we demonstrated that baker's yeast strains with high rates of mtDNA fragments migrating to the nucleus (yme1-1 mutant) exhibit short chronological life spans (CLS). The yeast CLS is determined by the survival of non-dividing cell populations. Here, we show that lack of the non-homologous-end-joining enzyme DNA ligase IV (DNL4) can rescue the short CLS of the yme1-1 mutant. In fission yeast, DNA ligase IV has been shown to be required for the capture of mtDNA fragments during the repair of double-stranded DNA breaks in nuclear DNA. In further analyses using pulse field gel and 2D gel electrophoresis we demonstrate that linear mtDNA fragments with likely nuclear localization accumulate in the yme1-1 mutant. The accumulation of the linear mtDNA fragments in the yme1-1 mutant is suppressed when Dnl4 is absent. We propose that the linear nuclear mtDNA fragments accelerate the aging process in the yme1-1 mutant cells by possibly affecting nuclear processes including DNA replication, recombination, and repair as well as transcription of nuclear genes. We speculate further that Dnl4 protein has besides its function as a ligase also a role in DNA protection. Dnl4 protein may stabilize the linear mtDNA fragments in the nucleus by binding to their physical ends. In the absence of Dnl4 protein the linear fragments are therefore unprotected and possibly degraded by nuclear nucleases.

Regulation of longevity by regulator of G-protein signaling protein, Loco.

Regulator of G-protein signaling (RGS) proteins contribute to G-protein signaling pathways as activators or repressors with GTPase-activating protein (GAP) activity. To characterize whether regulation of RGS proteins influences longevity in several species, we measured stress responses and lifespan of RGS-overexpressing and RGS-lacking mutants. Reduced expression of Loco, a RGS protein of Drosophila melanogaster, resulted in a longer lifespan for both male and female flies, also exhibiting stronger resistance to three different stressors (starvation, oxidation, and heat) and higher manganese-containing superoxide dismutase (MnSOD) activity. In addition, this reduction in Loco expression increased fat content and diminished cAMP levels. In contrast, overexpression of both genomic and cDNA loco gene significantly shortened the lifespan with weaker stress resistance and lower fat content. Deletion analysis of the Loco demonstrated that its RGS domain is required for the regulation of longevity. Consistently, when expression of RGS14, mammalian homologue of Loco, was reduced in rat fibroblast cells, the resistance to oxidative stress increased with higher MnSOD expression. The changes of yeast Rgs2 expression, which shares a conserved RGS domain with the fly Loco protein, also altered lifespan and stress resistance in Saccharomyces cerevisiae. Here, we provide the first evidence that RGS proteins with GAP activity affect both stress resistance and longevity in several species.

Association of the yeast DNA helicase Pif1p with mitochondrial membranes and mitochondrial DNA.

Previously we demonstrated that the mitochondrial form of the yeast Pif1p DNA helicase, which we found to be attached to mitochondrial DNA (mtDNA), is required for the maintenance of mtDNA under genotoxic stress conditions. Here, we demonstrated that mitochondrial Pif1p is exclusively bound to mitochondrial membranes and part of an about 900kDa protein complex. Pif1p might be incorporated into this complex immediately after its translocation from the cytoplasm into the mitochondrial matrix. Pif1p as well as the mitochondrial DNA polymerase Mip1p could not be released from the mitochondrial membranes by digesting mtDNA with restriction enzymes in permeabilized mitochondria. In contrast, restriction enzyme-digested mtDNA fragments that were covered by the histone-like protein Abf2p were efficiently released from the permeabilized mitochondria. We propose that Pif1p as well as Mip1p are not only bound to mtDNA but also to the inner mitochondrial membrane either directly or indirectly via a protein complex. We also found that in the absence of mtDNA the total amount of cellular Pif1p is highly reduced.