Aged Cardiomyocytes Research Articles

Lysophosphatidylethanolamine improves diastolic dysfunction by alleviating mitochondrial injury in the aging heart

Diastolic dysfunction in aging mice is linked to mitochondrial abnormalities, including mitochondrial morphology disorders and decreases in membrane potential. Studies also show that aberrant mitochondrial lipid metabolism impairs mitochondrial function in aging cardiomyocytes. Our lipidomic analysis revealed that phosphatidylethanolamine (PE) levels were significantly decreased in aging myocardial mitochondria. Here, we investigated whether reduction in PE levels in myocardial mitochondria contributes to mitochondrial injury as well as HFpEF pathogenesis, and whether modulation of PE levels could ameliorate aging-induced HFpEF. Echocardiography was used to assess cardiac diastolic function in adult and aging mice treated with lysophosphatidylethanolamine (LPE) or saline. Mitochondrial morphologies from tissue samples were evaluated by transmission electron microscopy (TEM), while mitochondrial membrane potential and reactive oxygen species (ROS) levels were assessed using JC-1, MitoSOX and DCFH-DA detection assays. We performed GO enrichment analysis between adult and aging mice and discovered significant enrichment in transcriptional programs associated with mitochondria and lipid metabolism. Also, mitochondrial PE levels were significantly decreased in aging cardiomyocytes. Treatment with LPE significantly enhanced PE content in aging mice and improved the structure of mitochondria in cardiac cells. Also, LPE treatment protected against aging-induced deterioration of mitochondrial injury, as evidenced by increased mitochondrial membrane potential and decreased mitochondrial ROS. Furthermore, treatment with LPE alleviated severe diastolic dysfunction in aging mice. Taken together, our results suggest that LPE treatment enhances PE levels in mitochondria and ameliorates aging-induced diastolic dysfunction in mice through a mechanism involving improved mitochondrial structure and function.

LncRNA UCA1 enhances NRF2 expression through the m6A pathway to mitigate oxidative stress and ferroptosis in aging cardiomyocytes.

To explore the regulatory mechanism of lncRNA UCA1 and NRF2 in cardiomyocyte aging. In this study, we explored how lncRNA UCA1 regulates NRF2 and its effect on cardiomyocyte aging. H9c2 cardiomyocytes were cultured and treated with H2O2 to simulate cardiomyocyte aging in vitro. The expression levels of lncRNA UCA1 and NRF2 in cells were detected using qRT-PCR. Cell viability was assessed using the CCK8 assay, and cell aging was detected via Sa-β-gal staining. The levels of oxidative stress markers (SOD, MDA, ROS) and the expressions of ferroptosis-related proteins (ACSL4, TFR1, FTH1, GPX4) were measured. The regulatory mechanism between UCA1 and NRF2 was investigated using RIP-qPCR. Additionally, changes in m6A modification levels and the expression of m6A modification-related proteins in cells after UCA1 overexpression were analyzed by western blot. Our results indicate that H2O2 treatment significantly downregulated the expression of lncRNA UCA1 and NRF2. UCA1 overexpression promoted H9c2 cell proliferation, inhibited cell aging, increased SOD activity and the expression of FTH1 and GPX4 proteins, and decreased MDA and ROS content as well as ACSL4 and TFR1 protein expression. RIP-qPCR verified that UCA1 can promote the expression of NRF2 in cells. Overexpression of UCA1 significantly increased the expression of the demethylase FTO, leading to a reduction in m6A modification levels. Furthermore, there was significant enrichment between FTO and NRF2, and overexpression of FTO improved the expression of NRF2 protein in cells. Taken together, lncRNA UCA1 inhibits oxidative stress and ferroptosis, thereby preventing cardiomyocyte aging. This protective effect is likely mediated by increasing the expression of demethylase FTO and reducing m6A modification, which promotes the expression of NRF2.

The mechanism by which piR-000699 targets SLC39A14 regulates ferroptosis in aging myocardial ischemia/reperfusion injury.

Myocardial ischemia/reperfusion (I/R) injury is a classic type of cardiovascular disease characterized by injury to cardiomyocytes leading to different types of cell death. The degree of irreversible myocardial damage is closely related to age, and ferroptosis is involved in cardiomyocyte damage. However, the mechanisms underlying ferroptosis regulation in aging myocardial I/R injury are still unclear. The present study aims to explore the underlying mechanism of piRNA regulation in ferroptosis. Using left anterior descending coronary artery ligation in an aging rat model and a D-galactose-induced rat cardiomyocyte line (H9C2) to construct an aging cardiomyocyte model, we investigate whether ferroptosis occurs after reperfusion injury in vitro and in vivo. This study focuses on the upregulation of piR-000699 after hypoxia/reoxygenation treatment in aging cardiomyocytes by observing hypoxia/reoxygenation (H/R) injury indicators and ferroptosis-related indicators and clarifying the role of piR-000699 in H/R injury caused by ferroptosis in aging cardiomyocytes. Bioinformatics analysis reveals that SLC39A14 is a gene that binds to piR-000699. Our data show that ferroptosis plays an important role in I/R injury both in vivo and in vitro. Furthermore, the results show the potential role of piR-000699 in regulating SLC39A14 in ferroptosis in aging cardiomyocytes under hypoxia/reoxygenation conditions. Together, our results reveal that the mechanism by which piR-000699 binds to SLC39A14 regulates ferroptosis in aging myocardial I/R injury.

A small molecule macrophage migration inhibitory factor agonist ameliorates age-related myocardial intolerance to ischemia-reperfusion insults via metabolic regulation

Macrophage migration inhibitory factor (MIF) is an innate cytokine that regulates both inflammatory and homeostatic responses. MIF is expressed by cardiomyocytes, where it exerts a protective action against ischemia-reperfusion (I/R) injury by activating AMP-activated protein kinase (AMPK). This effect is attenuated in the senescent heart due to an intrinsic, age-related reduction in MIF expression. We hypothesized that treating the aged heart with the small molecule MIF agonist (MIF20) can reinforce protective MIF signaling in cardiomyocytes, leading to a beneficial effect against I/R stress. The administration of MIF20 at the onset of reperfusion was found to not only decrease myocardial infarct size but also preserves systolic function in the aged heart. Protection from I/R injury was reduced in mice with cardiomyocyte-specific Mif deletion, consistent with the mechanism of action of MIF20 to allosterically increase MIF affinity for its cognate receptor CD74. We further found MIF20 to contribute to the maintenance of mitochondrial fitness and to preserve the contractile properties of aged cardiomyocytes under hypoxia/reoxygenation. MIF20 augments protective metabolic responses by reducing the NADH/NAD ratio, leading to a decrease in the accumulation of reactive oxygen species (ROS) in the aged myocardium under I/R stress. We also identify alterations in the expression levels of the downstream effectors PDK4 and LCAD, which participate in the remodeling of the cardiac metabolic profile. Data from this study demonstrates that pharmacologic augmentation of MIF signaling provides beneficial homeostatic actions on senescent myocardium under I/R stress.

Aging Triggers Mitochondrial Dysfunction in Mice.

Direct analysis of isolated mitochondria from old mice enables a better understanding of heart senescence dysfunction. Despite a well-defined senescent phenotype in cardiomyocytes, the mitochondrial state in aged cardiomyocytes is still unclear. Here, we report data about mitochondrial function in old mice. Isolated cardiomyocytes' mitochondria were obtained by differential centrifugation from old and young mice hearts to perform functional analyses of mitochondrial O2 consumption, transmembrane potential, ROS formation, ATP production, and swelling. Our results show that mitochondria from old mouse hearts have reduced oxygen consumption during the phosphorylative states of complexes I and II. Additionally, these mitochondria produced more ROS and less ATP than those of young hearts. Mitochondria from old hearts also showed a depolarized membrane potential than mitochondria from young hearts and, as expected, a greater electron leak. Our results indicate that mitochondria from senescent cardiomyocytes are less efficient in O2 consumption, generating more ROS and producing less ATP. Furthermore, the phosphorylative state of complexes I and II presents a functional defect, contributing to greater leakage of protons and ROS production that can be harmful to the cell.

An interferon gamma response signature links myocardial aging and immunosenescence.

Aging entails profound immunological transformations that can impact myocardial homeostasis and predispose to heart failure. However, preclinical research in the immune-cardiology field is mostly conducted in young healthy animals, which potentially weakens its translational relevance. Herein, we sought to investigate how the aging T-cell compartment associates with changes in myocardial cell biology in aged mice. We phenotyped the antigen-experienced effector/memory T cells purified from heart-draining lymph nodes of 2-, 6-, 12-, and 18-month-old C57BL/6J mice using single-cell RNA/T cell receptor sequencing. Simultaneously, we profiled all non-cardiomyocyte cell subsets purified from 2- to 18-month-old hearts and integrated our data with publicly available cardiomyocyte single-cell sequencing datasets. Some of these findings were confirmed at the protein level by flow cytometry. With aging, the heart-draining lymph node and myocardial T cells underwent clonal expansion and exhibited an up-regulated pro-inflammatory transcription signature, marked by an increased interferon-γ (IFN-γ) production. In parallel, all major myocardial cell populations showed increased IFN-γ responsive signature with aging. In the aged cardiomyocytes, a stronger IFN-γ response signature was paralleled by the dampening of expression levels of transcripts related to most metabolic pathways, especially oxidative phosphorylation. Likewise, induced pluripotent stem cells-derived cardiomyocytes exposed to chronic, low grade IFN-γ treatment showed a similar inhibition of metabolic activity. By investigating the paired age-related alterations in the T cells found in the heart and its draining lymph nodes, we provide evidence for increased myocardial IFN-γ signaling with age, which is associated with inflammatory and metabolic shifts typically seen in heart failure.

Cardiomyocyte Pdk4 response is associated with metabolic maladaptation in aging.

Ischemic heart disease (IHD) is the leading cause of death, with age range being the primary factor for development. The mechanisms by which aging increases vulnerability to ischemic insult are not well understood. We aim to use single-cell RNA sequencing to discover transcriptional differences in various cell types between aged and young mice, which may contribute to aged-related vulnerability to ischemic insult. Utilizing 10× Genomics Single-Cell RNA sequencing, we were able to complete bioinformatic analysis to identity novel differential gene expression. During the analysis of our collected samples, we detected Pyruvate Dehydrogenase Kinase 4 (Pdk4) expression to be remarkably differentially expressed. Particularly in cardiomyocyte cell populations, Pdk4 was found to be significantly upregulated in the young mouse population compared to the aged mice under ischemic/reperfusion conditions. Pdk4 is responsible for inhibiting the enzyme pyruvate dehydrogenase, resulting in the regulation of glucose metabolism. Due to decreased Pdk4 expression in aged cardiomyocytes, there may be an increased reliance on glucose oxidization for energy. Through biochemical metabolomics analysis, it was observed that there is a greater abundance of pyruvate in young hearts in contrast to their aged counterparts, indicating less glycolytic activity. We believe that Pdk4 response provides valuable insight towards mechanisms that allow for the young heart to handle ischemic insult stress more effectively than the aged heart.

ALKBH5 alleviates hypoxia postconditioning injury in D-galactose-induced senescent cardiomyocytes by regulating STAT3.

Ischemic postconditioning (I/Post) reduces ischemia/reperfusion (I/R) injury by activating endogenous cardioprotection mechanisms, such as the JAK/STAT3 and PI3K/Akt pathways, which offer a traditional approach to myocardial protection. According to the previous study, cardioprotection by I/Post may lost in aged mice, and in our previous research, hypoxic postconditioning (H/Post) lacked a protective effect in senescent cardiomyocytes, which was associated with low expression of long noncoding RNA (lncRNA) H19. The N6-methyladenosine (m6A) modification is a dynamic and reversible process that has been confirmed plays a role in cardiovascular diseases. However, the mechanisms of m6A modification in myocardial I/Post remains to be explored. Neonatal cardiomyocytes were isolated from 2-day-old Sprague Dawley rats and senescence was induced by D-galactose, followed by stimulation of hypoxia-reoxygenation (H/R) and H/Post. Hypoxic injury was evaluated by cell viability and the Bcl-2/Bax protein ratio. Total m6A levels were measured using a colorimetric m6A RNA Methylation Quantification Kit and the m6A modified and differentially expressed mRNA was determined by methylated RNA immunoprecipitation (MeRIP). We found that H/Post increased m6A methylation and decreased RNA mA demethylase alkB homolog 5 (ALKBH5) expression in aged cardiomyocytes. Furthermore, ALKBH5 knockdown exacerbated injury following H/Post in senescent cardiomyocytes. Additionally, ALKBH5 regulated STAT3 expression by mediating its m6A modification and lncRNA H19/miR-124-3p. ALKBH5 also alleviated the H/Post injury induced by the low expression of STAT3 in senescent cardiomyocytes.

Inner mitochondrial membrane structure and fusion dynamics are altered in senescent human iPSC-derived and primary rat cardiomyocytes

Dysfunction of the aging heart is a major cause of death in the human population. Amongst other tasks, mitochondria are pivotal to supply the working heart with ATP. The mitochondrial inner membrane (IMM) ultrastructure is tailored to meet these demands and to provide nano-compartments for specific tasks. Thus, function and morphology are closely coupled. Senescent cardiomyocytes from the mouse heart display alterations of the inner mitochondrial membrane. To study the relation between inner mitochondrial membrane architecture, dynamics and function is hardly possible in living organisms. Here, we present two cardiomyocyte senescence cell models that allow in cellular studies of mitochondrial performance. We show that doxorubicin treatment transforms human iPSC-derived cardiomyocytes and rat neonatal cardiomyocytes in an aged phenotype. The treated cardiomyocytes display double-strand breaks in the nDNA, have β-galactosidase activity, possess enlarged nuclei, and show p21 upregulation. Most importantly, they also display a compromised inner mitochondrial structure. This prompted us to test whether the dynamics of the inner membrane was also altered. We found that the exchange of IMM components after organelle fusion was faster in doxorubicin-treated cells than in control cells, with no change in mitochondrial fusion dynamics at the meso-scale. Such altered IMM morphology and dynamics may have important implications for local OXPHOS protein organization, exchange of damaged components, and eventually the mitochondrial bioenergetics function of the aged cardiomyocyte.

Abstract 10387: Macrophage Migration Inhibitory Factor Maintains Mitochondrial Integrity in Aging During Ischemic Insults

Introduction: The expression and secretion of cardiac MIF (macrophage migration inhibitory factor) are downregulated in aging. A small molecule MIF agonist, MIF20 can increase the binding affinity of MIF with CD74 receptor to modulate AMPK signaling. However, the pharmacological mechanisms by which MIF20 triggers MIF-AMPK signaling cascade to benefit heart in response to ischemia and reperfusion (I/R) stress remain unknown. Hypothesis: MIF20 rescues an impaired MIF-AMPK signaling in aging through augmenting the interaction between MIF and CD74 receptor to maintain mitochondrial integrity of aged heart under ischemic stress. Methods: Young (3-5 months) and aged (21-24 months) C57BL/6J mice were subjected to 45 min of ligation of LAD (left anterior descending coronary artery) and injection of MIF20 (0.15 μg/kg, i.v.) 5 min before 24 hr of reperfusion with release of LAD. Echocardiography measures cardiac functions, and TTC/Evans blue staining for determination of myocardial infarction size. Seahorse XF Analyzer for mitochondrial respiration functions of isolated cardiomyocytes. Results: The echocardiography data showed that I/R caused cardiac systolic dysfunctions but not effect on cardiac diastolic functions of both young and aged C57BL/6J mice. There were no differences between male and female groups. Administration of MIF20 significantly ameliorated I/R-induced cardiac dysfunctions in both young and aged mice, while more improvement observed in aged versus young mice. MIF20 treatment reduced myocardial infarct size caused by I/R in both young and aged mice, but the reduction extent of aged hearts was higher than that of young hearts (p<0.05). Mitochondrial respiration rates of isolated cardiomyocytes as indicted by ATP production were impaired by I/R versus sham operations in both young and aged hearts. Intriguingly, MIF20 treatment significantly rescued the impaired mitochondrial functions caused by I/R in both young and aged cardiomyocytes. Moreover, the mitochondrial functions of young and aged cardiomyocytes were improved to a similar level under I/R with MIF20. Conclusions: MIF agonist MIF20 rescues the impaired energy metabolism in aged heart. The mitochondrial integrity under I/R by MIF20 could limit ischemic insults in aging.

Abstract 10461: Single-Cell Transcriptional Analysis of Altered Cardiomyocyte Pdk4 as Indicator of Ischemic Stress Vulnerability in Aging

Introduction: The mechanisms by which aging increases vulnerability to ischemic insult are not well understood. Single-Cell RNA sequencing (scRNA seq) was employed to characterize transcriptional differences in various cell types between aged and young mice which may contribute to aged-related vulnerability to ischemic insult. Pyruvate Dehydrogenase Kinase 4 (Pdk4) has been shown to impact capacity for fatty-acid oxidation (FAO) and consequently adaption to metabolic alterations of ischemic insult. Hypothesis: Alteration of Pdk4 expression levels is an adaptive response transcriptionally under pathological stress to maintain metabolic homeostasis during ischemia and reperfusion (I/R). Methods: Young (3-5 months) and aged (24-26 months) C57BL/6J mice were subjected to in vivo regional 45 min of ischemia and 24 hr of reperfusion (I/R) or sham operations. Bioinformatic analysis was done using Seurat integration vignettes allowing for cell type identification and differential expression testing. Observing genetic expression profiles by cell types. Substrate metabolism was determined with working heart system and Seahorse XF Analyzer. Results: Pdk4 was profoundly found in cardiomyocytes versus fibroblasts, endothelial cells and macrophages of both young and aged hearts. Interestingly, I/R stress triggered Pdk4 mRNA expression of young but not aged cardiomyocytes. Moreover, there is a relative higher Pdk4 level in aged versus young cardiomyocytes under sham operations, indicating Pdk4 mRNA respond to pathological stress. The ex vivo working heart perfusion data showed that young versus aged hearts exhibited higher FAO in response to I/R. Moreover, Seahorse XF Analyzer showed a higher glycolysis rate in young versus aged cardiomyocytes during I/R. It suggests that an impaired cardiomyocyte Pdk4 mRNA response occurred in aging under I/R stress conditions, this could result in cardiac maladaptive metabolic alterations in aging during pathological stress. Conclusions: Alterations in Pdk4 levels of cardiomyocytes regulates cardiac adaptive metabolism in response to pathological stress. This adaptive regulation of cardiomyocyte Pdk4 is impaired in aging that leads to more vulnerability of aged versus young hearts to ischemic insult.

Exogenous hydrogen sulfide inhibits the senescence of cardiomyocytes through modulating mitophagy in rats

Hydrogen sulfide (H2S), a gaseous molecule, has been shown to be involved in the regulation of body pathophysiological processes. Aging is related to structural and functional alterations within the heart. There is evidence that diminished mitophagy accelerates the aging process. Studies in recent years have revealed that plasma levels of H2S in humans and old rats decrease with age, and H2S acts as a cytoprotective mediator in the aging process. However, it is unclear whether H2S can delay the senescence of cardiomyocytes by regulating mitophagy. Our present results showed that exogenous H2S inhibited mitochondrial damage, oxidative stress and cell apoptosis, and enhanced mitophagy through upregulating the SIRT1-PINK1-parkin pathway in myocardial tissues of aged rats and cultured aged cardiomyocytes. Furthermore, the effect of exogenous H2S on the above indicators was the same as that of SRT1720 (a SIRT1 agonist) and kinetin (a PINK1 activator). Our findings suggest that exogenous H2S inhibits the senescence of cardiomyocytes by increasing mitophagy via upregulation of the SIRT1-PINK1-parkin pathway in rats.

Single-nucleus transcriptomics reveals a gatekeeper role for FOXP1 in primate cardiac aging.

Aging poses a major risk factor for cardiovascular diseases, the leading cause of death in the aged population. However, the cell type-specific changes underlying cardiac aging are far from being clear. Here, we performed single-nucleus RNA-sequencing analysis of left ventricles from young and aged cynomolgus monkeys to define cell composition changes and transcriptomic alterations across different cell types associated with age. We found that aged cardiomyocytes underwent a dramatic loss in cell numbers and profound fluctuations in transcriptional profiles. Via transcription regulatory network analysis, we identified FOXP1, a core transcription factor in organ development, as a key downregulated factor in aged cardiomyocytes, concomitant with the dysregulation of FOXP1 target genes associated with heart function and cardiac diseases. Consistently, the deficiency of FOXP1 led to hypertrophic and senescent phenotypes in human embryonic stem cell-derived cardiomyocytes. Altogether, our findings depict the cellular and molecular landscape of ventricular aging at the single-cell resolution, and identify drivers for primate cardiac aging and potential targets for intervention against cardiac aging and associated diseases.

Potential Role of Exercise in Regulating YAP and TAZ During Cardiomyocytes Aging.

Adaptation of cardiac muscle to regular exercise results in morphological and structural changes known as physiological cardiac hypertrophy, to which the Hippo signaling pathway might have contributed. Two major terminal effectors in the Hippo signaling pathway are Yes-associated protein (YAP) and its homolog transcriptional coactivator with PDZ-binding motif (TAZ). The latest studies have reported the role of YAP and TAZ in different life stages, such as in fetal, neonatal, and adult hearts. Their regulation might involve several mechanisms and effectors. One of the possible coregulators is exercise. Exercise plays a role in cardiomyocyte hypertrophic changes during different stages of life, including in aged hearts. YAP/TAZ signaling pathway has a role in physiological cardiac hypertrophy induced by exercise and is associated with cardiac remodelling. Thus, it can be believed that exercise has roles in activating the signaling pathway of YAP and TAZ in aged cardiomyocytes. However, the studies regarding the roles of YAP and TAZ during cardiomyocyte aging are limited. The primary purpose of this review is to explore the response of cardiovascular aging to exercise via signaling pathway of YAP and TAZ.

Exercise Training Ameliorates Cardiac Aging Of Mice Through Activating Apelin/apj System

PURPOSE: Apelin/APJ system is known to improve cardiovascular disease. Exercise training has been associated with benefits against cardiac diseases and aging associated disorders. Our previous research showed that the serum Apelin levels decreased with age and exogenous Apelin intervention or overexpression of its receptor APJ could reverse the aging phenotype in D-galactose-induced aging cardiomyocytes. Herein we investigated the role Apelin/APJ system played in exercise training ameliorates cardiac aging. METHODS: The aging heart was simulated in 24 weeks-old senescence-accelerated mice. Mice were subjected to 6 weeks treadmill training, and treated with or without intraperitoneal injection of the apelin receptor antagonist - F13A 75ug/kg per day during training. The effects of treadmill training on circulating Apelin, myocardial Apelin / APJ, cardiac aging phenotype and mitochondrial autophagy were evaluated. RESULTS: Exercise training reduced weight loss, myocardial fibrosis and cardiac expression of aging biomarkers (p16, p53 and p21) in aged mice. In addition, exercise training increased the expression of Apelin and APJ in cardiomyocytes and maintained mitochondrial homeostasis, as evidenced by expression of mitochondrial related proteins (PGC-1α, CPT1A, PINK1, Parkin, Drp-1 and fis-1). The cardioprotective effects of exercise training were weakened by inhibition of the Apelin/APJ system. CONCLUSIONS: Taken together, our research showed that exercise training ameliorated phenotype of cardiac aging and disruption of mitochondrial homeostasis through activating the Apelin/APJ system. Hence, pharmacological activation of the Apelin/APJ system may prove to be a promising prevention against aging related heart disease especially in subjects with intolerance of exercise.

Wnt 3a Protects Myocardial Injury in Elderly Acute Myocardial Infarction by Inhibiting Serum Cystatin C/ROS-Induced Mitochondrial Damage

Aging represents an independent risk factor affecting the poor prognosis of patients with acute myocardial infarction (AMI). This present research aimed to explore the molecular mechanism of myocardial injury in elderly AMI by animals and cells experiment. Our previous clinical study found the serum Cystatin C (Cys-C) increased in the elderly AMI population, while the mechanism underlying high Cys-C induced myocardial injury of AMI remains unclear. In the in-vitro study, we confirmed that Wnt/β-catenin could significantly reduce the expression of cytoplasmic Cys-C through transnuclear action, and highly attenuate the occurrence of mitochondrial oxidative stress injury induced via Cys-C/reactive oxygen species (ROS). Furthermore, the addition of exogenous Wnt3a and inhibition of Cys-C expression could effectively inhibit mitochondrial oxidative stress injury and relieve the acute myocardial hypoxia injury. These results indicate that Cys-C exerted damaging effects on the hypoxic aging cardiomyocyte through the ROS/mitochondrial signaling pathway. Inhibition of this pathway effectively reduced the apoptosis of aging cardiomyocytes. In the in-vivo study, we also explored the function of the Wnt/Cys-C pathway on the ischemic infarction heart. We confirmed that Wnt/β-catenin served as the upstream protective protein of this pathway, and the promotion of this pathway improved the cardiac structure and function of the elderly AMI mice effectively.

Characterization of early age-associated remodelling of cAMP-phosphodiesterases and β-adrenergic regulation of excitation–contraction coupling

The most common form of heart failure (HF) in the elderly is HF with preserved ejection fraction (HFpEF), characterized by impaired filling and relaxation of the heart. Ageing is accompanied by an activation of the sympathetic nervous system and β-adrenergic (β-AR) desensitization, followed by reduced cardiomyocyte contractility, yet little is known about the early involved mechanisms. We aimed to study cAMP-phosphodiesterase (PDE) signaling changes upon β-AR stimulation in an early ageing model and its contribution to excitation–contraction coupling. Wistar rats at 3 and 15 months of age were used as young vs. early aged rat model. Ventricular myocytes were isolated and expression of senescent marker genes (p15/16/p21) and phosphodiesterases (PDEs) was assessed by qPCR. Total cAMP-PDE activity as well as PDE3 and PDE4 specific activities were measured by radioimmunoassay. Sarcomere shortening and Ca2+ transients were assessed in field-stimulated (1 Hz) cardiomyocytes loaded with the Ca2+ dye, Fura 2 (1 μM). In parallel, cells were infected with adenoviruses encoding the cytosolic cAMP FRET sensor, EpacSH187. Concentration-response curves to the β-AR agonist isoproterenol (Iso) were obtained. The early ageing phenotype of our model was demonstrated through a physiological increase in heart weight that was back to normal when divided by tibia length and through the absence of detectable p15/16/p21 levels. The expression of PDE2A, PDE3A, PDE4A, PDE4B and PDE4D at the transcript level was not modified in young vs. aged cardiomyocytes ( n = 7/group). Total PDE activity, as well as specific PDE3 and PDE4 activities tended to be increased in cardiomyocytes from aged vs. young rats, although it did not reach statistical significance ( n = 8/group). This was accompanied by a significantly diminished efficacy of Iso to increase cytosolic cAMP levels and Ca2+ transient amplitude in cardiomyocytes from old vs. young rats ( n = 5–9/group, P < 0.05, extra sum-of-squares F test). However, basal and Iso-stimulated sarcomere shortening were similar in cardiomyocytes from aged vs. young rats. Our data show that cardiomyocyte contractility is preserved in 15 months old rats despite decreased Ca2+ transients and suggest a precocious alteration of cAMP-PDEs at this early ageing stage. Further experiments are being pursued to depict the mechanisms underlying depressed Ca2+ transients and increased myofilament Ca2 + sensitivity in early aging.

Ischemic Postconditioning Protects against Aged Myocardial Ischemia/Reperfusion Injury by Transcriptional and Epigenetic Regulation of miR-181a-2-3p.

Ischemic postconditioning (IPostC) has been proposed as a strategy to mitigate the risk of ischemia/reperfusion (I/R) injury, and autophagy is involved in I/R-induced aged myocardial injury, while the underlying mechanism of IPostC-regulated autophagy is unknown. Here, we implemented miRNA sequencing analysis in aged cardiomyocytes to identify a novel miR-181a-2-3p after HPostC, which inhibits autophagy by targeting AMBRA1 in aged myocardium to protect I/R-induced aged myocardial injury. Mechanistically, we identified that IPostC can induce DNA hypomethylation and H3K14 hyperacetylation of miR-181a-2-3p promoter due to the decreased binding of DNMT3b and HDAC2 at its promoter, which contributes to enhancing the expression of miR-181a-2-3p. More importantly, cooperation of DNMT3b and HDAC2 inhibits the binding of c-Myc at the miR-181a-2-3p promoter in aged cardiomyocytes. In summary, IPostC attenuates I/R-induced aged myocardial injury through upregulating miR-181a-2-3p expression, which is an attribute to transcriptional and epigenetic regulation of its promoter. Our data indicate that miR-181a-2-3p may be a potential therapeutic target against I/R injury in aged myocardium.

Bmi-1-RING1B prevents GATA4-dependent senescence-associated pathological cardiac hypertrophy by promoting autophagic degradation of GATA4.

AimsSenescence‐associated pathological cardiac hypertrophy (SA‐PCH) is associated with upregulation of foetal genes, fibrosis, senescence‐associated secretory phenotype (SASP), cardiac dysfunction and increased morbidity and mortality. Therefore, we conducted experiments to investigate whether GATA4 accumulation induces SA‐PCH, and whether Bmi‐1‐RING1B promotes GATA4 ubiquitination and its selective autophagic degradation to prevent SA‐PCH.Methods and results Bmi‐1‐deficient (Bmi‐1−/− ), transgenic Bmi‐1 overexpressing (Bmi‐1Tg ) and wild‐type (WT) mice were infused with angiotensin II (Ang II) to stimulate the development of SA‐PCH. Through bioinformatics analysis with RNA sequencing data from cardiac tissues, we found that Bmi‐1‐RING1B and autophagy are negatively related to SA‐PCH. Bmi‐1 deficiency promoted GATA4‐dependent SA‐PCH by increasing GATA4 protein and hypertrophy‐related molecules transcribed by GATA4 such as ANP and BNP. Bmi‐1 deficiency stimulated NF‐κB‐p65‐dependent SASP, leading to cardiac dysfunction, cardiomyocyte hypertrophy and senescence. Bmi‐1 overexpression repressed GATA4‐dependent SA‐PCH. GATA4 degraded by Bmi‐1 was mainly dependent on autophagy rather than proteasome. In human myocardium, p16 positively correlated with ANP and GATA4 and negatively correlated with LC3B, Bmi‐1 and RING1B; GATA4 positively correlated with p62 and negatively correlated with Bmi‐1 and LC3B. With increased p16 protein levels, ANP‐, BNP‐ and GATA4‐positive cells or areas increased; however, LC3B‐positive cells or areas decreased in human myocardium. GATA4 is ubiquitinated after combining with Bmi‐1‐RING1B, which is then recognised by p62, is translocated to autophagosomes to form autophagolysosomes and degraded. Downregulated GATA4 ameliorated SA‐PCH and cardiac dysfunction by reducing GATA4‐dependent hypertrophy and SASP‐related molecules. Bmi‐1 combined with RING1B (residues 1–179) and C‐terminus of GATA4 (residues 206–443 including zinc finger domains) through residues 1–95, including a RING‐HC‐finger. RING1B combined with C‐terminus of GATA4 through the C‐terminus (residues 180–336). Adeno‐associated viral vector serotype 9 (AAV9)‐cytomegalovirus (CMV)‐Bmi‐1‐RING1B treatment significantly attenuated GATA4‐dependent SA‐PCH through promoting GATA4 autophagic degradation.ConclusionsBmi‐1‐RING1B maintained cardiac function and prevented SA‐PCH by promoting selective autophagy for degrading GATA4.Translational perspectiveAAV9‐CMV‐Bmi‐1‐RING1B could be used for translational gene therapy to ubiquitinate GATA4 and prevent GATA4‐dependent SA‐PCH. Also, the combined domains between Bmi‐1‐RING1B and GATA4 in aging cardiomyocytes could be therapeutic targets for identifying stapled peptides in clinical applications to promote the combination of Bmi‐1‐RING1B with GATA4 and the ubiquitination of GATA4 to prevent SA‐PCH and heart failure. We found that degradation of cardiac GATA4 by Bmi‐1 was mainly dependent on autophagy rather than proteasome, and autophagy agonists metformin and rapamycin could ameliorate the SA‐PCH, suggesting that activation of autophagy with metformin or rapamycin could also be a promising method to prevent SA‐PCH.

Ageing Increases Cardiac Electrical Remodelling in Rats and Mice via NOX4/ROS/CaMKII-Mediated Calcium Signalling.

Objective Ageing is one of the risk factors associated with cardiovascular diseases including cardiac arrhythmias and heart failure. Ageing-related cardiac dysfunction involves a complicated pathophysiological progress. Abnormal membrane voltage and Ca2+ dynamics in aged cardiomyocytes contribute to ageing-related arrhythmias. However, its underlying mechanisms have not been well clarified. Methods Young and old rats or mice were included in this study. Cardiac electrophysiological properties and functions were assessed by ECG, echocardiography, and ex vivo heart voltage and Ca2+ optical mapping. Proteomics, phosphor-proteomics, Western blotting, Masson staining, and ROS measurement were used to investigate the underlying mechanisms. Results Ageing increased the incidence of cardiac hypertrophy and fibrosis in rats. Moreover, ageing increased the occurrence of ventricular tachycardia or ventricular fibrillation induced by rapid pacing and during isoprenaline (ISO) (1 mg/kg i.p.) challenge in mice in vivo. Optical mapping with dual dyes (membrane voltage (Vm) dye and intracellular Ca2+ dye) simultaneously recording revealed that ageing increased the action potential duration (APD) and Ca2+ transient duration (CaTD) and slowed the ventricular conduction with the Langendorff-perfused mouse heart. More importantly, ageing increased the ISO-induced (1 μM) changes of APD (ΔAPD80) and CaTD (ΔCaTD50). Ageing also delayed the decay of Ca2+ transient by extending the decay time constant from 30% to 90% (τ30−90). In addition, ageing decreased the Vm/Ca2+ latency which represented the coupling of Vm/Ca2+ including between the midpoint of AP depolarization and Ca2+ upstroke, peak transmembrane voltage and peak cytosolic calcium, and time to 50% voltage repolarization and extrusion of cytosolic calcium. Optical mapping also revealed that ageing increased the ISO-induced arrhythmia incidence and occurrence of the excitation rotor. Proteomics and phosphor-proteomics assays from rat hearts demonstrated ageing-induced protein and phosphor-protein changes, suggesting that CaMKII was involved in ageing-induced change. Ageing increased the level of ROS and the expression of NOX4, oxidative CaMKII (ox-CaMKII), phosphorated CaMKII (p-CaMKII), and periostin. Conclusion Ageing accelerates cardiac remodelling and increases the susceptibility to ventricular arrhythmias through NOX4/ROS/CaMKII pathway-mediated abnormal membrane voltage and intracellular Ca2+ handling and Vm/Ca2+ coupling.