Andreas S. Ivessa

Background/Objectives: Cardiomyopathy (CM) is a leading cause of morbidity and mortality in Duchenne muscular dystrophy (DMD) patients. Ferroptosis, an iron-dependent form of cell death characterized by lipid peroxidation, is implicated in various cardiovascular diseases. However, the role of ferroptosis in DMD-CM remains unexplored. Methods: Here, we used dystrophin and utrophin double-knockout (mdx:utr^−/−) mice as a model that exhibits cardiac pathological phenotypes similar to those seen in DMD patients to investigate the potential role of ferroptosis. Results: We observed an increased level of iron deposition and lipid peroxidation in the hearts of mdx:utr^−/− mice. Live/Dead viability assays revealed that mdx:utr^−/− cardiomyocytes exhibited greater susceptibility to ferroptosis than WT cardiomyocytes both at baseline and upon exposure to ferroptosis inducers. We also used mdx:utr^−/− mice with a heterozygous sarcolipin (SLN) knockout background (sln^+/−) to investigate the effect of SLN reduction on ferroptosis susceptibility in DMD-CM. Notably, ferroptosis was significantly suppressed in cardiomyocytes from mdx:utr^−/−:sln^+/− mice (p < 0.01). Western blot analysis confirmed the upregulation of transferrin receptor 1 (TfR1) and 15-lipoxygenase-1 (15LOX1), along with the downregulation of heme oxygenase-1 (HMOX-1) and ferroptosis suppressor protein 1 (FSP1) in mdx:utr^−/− hearts, while glutathione peroxidase 4 (GPX4) levels remained unchanged. A similar pattern of alterations in ferroptosis-related biomarkers was observed in human heart samples from DMD patients compared to healthy controls. Conclusions: Our results provide direct evidence that ferroptosis contributes to the pathology of DMD-CM and suggest that reducing SLN expression and inhibiting ferroptosis may represent potential therapeutic strategies for this condition.

Aims: Doxorubicin (Dox) is an effective chemotherapeutic agent, but its clinical use is limited by cardiotoxicity. Cellular senescence contributes to Dox-induced cardiac dysfunction; however, the underlying molecular mechanism mediating the effect of senescence remains poorly understood. This study aimed to identify senescence-associated factors secreted from cardiomyocytes in Dox-treated hearts and define their functional significance in Dox-induced cardiotoxicity. Methods: Mice with cardiomyocyte-specific expression of the endoplasmic reticulum BioID secretome profiling system were used to identify Dox-induced secreted factors. Functional analyses were performed in neonatal rat ventricular myocytes (NRVMs). The effects of plasminogen activator inhibitor-1 (PAI-1) inhibition were evaluated in Dox-treated mice by assessing senescence markers, apoptotic responses, and cardiac structure and function. p21High-tdTomato reporter mice were used to examine the fate of senescent cardiomyocytes in vivo. Results: PAI-1 was identified as a major component of the senescence-associated secretory phenotype and was robustly upregulated in Dox-treated cardiomyocytes. In NRVMs, PAI-1 promoted senescence and maintained the senescent phenotype, in part by conferring resistance to apoptosis. Pharmacological inhibition of PAI-1 reduced senescence markers, enhanced apoptotic responses, and preserved cardiac structure and function in Dox-treated mice. Fate mapping analyses with p21High-tdTomato mice revealed that PAI-1 inhibition decreased the number of p21High senescent cardiomyocytes in Dox-treated hearts. Notably, PAI-1 inhibition did not attenuate Dox cytotoxicity in EO771 murine breast cancer cells. Conclusion: PAI-1 is a key mediator of Dox-induced cardiac dysfunction. PAI-1 inhibition shifts the fate of cardiomyocytes from senescence toward apoptosis and preserves cardiac structure and function without compromising the antitumor function of Dox, highlighting PAI-1 as a potential therapeutic target for chemotherapy-associated cardiotoxicity.

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.

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. 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. 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.

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-3β, was re-activated by both LC diets; however, the Fat-LC, but not the Pro-LC, diet exerted cardioprotection in GSK-3β cardiac-specific knockout mice. β-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.

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. Telomere length is maintained primarily through equilibrium between telomerase-mediated lengthening and the loss of telomeric sequence through the end-replication problem. In budding yeast Rap1 protein binds to telomeric TG repeat and negatively regulates telomerase recruitment in a dosage-dependent manner. In this paper we provide evidence suggesting an alternative Rap1-dependent telomere shortening mechanism in which binding of multiple Rap1 proteins mediates DNA break induction during DNA replication. This process does not involve recombination events; therefore, it is distinct from loop-mediated telomere trimming.

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.