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BACKGROUND: The mechanism of cardiac reverse remodeling (CRR) mediated by the left ventricular assist device remains unclear. This study aims to identify the specific cell type responsible for CRR and develop the therapeutic target that promotes CRR. The nuclei were extracted from the left ventricular tissue of 4 normal controls, 4 CRR patients, and 4 no cardiac reverse remodeling patients and then subjected to single-nucleus RNA sequencing for identifying key cell types responsible for CRR. Gene overexpression in transverse aortic constriction and dilated cardiomyopathy heart failure mouse model (C57BL/6J background) and pathological staining were performed to validate the results of single-nucleus RNA sequencing. RESULTS: Ten cell types were identified among 126 156 nuclei. Cardiomyocytes in CRR patients expressed higher levels of ATP5F1A than the other 2 groups. The macrophages in CRR patients expressed more anti-inflammatory genes and functioned in angiogenesis. Endothelial cells that elevated in no cardiac reverse remodeling patients were involved in the inflammatory response. Echocardiography showed that overexpressing ATP5F1A through cardiomyocyte-specific adenoassociated virus 9 demonstrated an ability to improve heart function and morphology. Pathological staining showed that overexpressing ATP5F1A could reduce fibrosis and cardiomyocyte size in the heart failure mouse model. CONCLUSIONS:' The present results of single-nucleus RNA sequencing and heart failure mouse model indicated that ATP5F1A could mediate CRR and supported the development of therapeutics for overexpressing ATP5F1A in promoting CRR. [ABSTRACT FROM AUTHOR]

Objective To investigate the regulatory effect of tRNA-derived small RNA (tsRNA) on ferroptosis in mouse cardiomyocytes and its mechanism. Methods RT-qPCR was used to measure the relative expression level of 5'tRF-LysCTT in the heart, liver, spleen, kidney, brain, and muscle of C57BL/6J mice aged 8 weeks. Primary cultured cardiomyocytes were divi-ded into groups A, B, C, and D: the cardiomyocytes in group A were cultured in a complete medium for 60 h; those in group B were cultured in a complete medium for 24 h, followed by starvation treatment for 12 h and H/R treatment for 24 h; those in groups C and D were transfected with antagomir-NC and antagomir-5'tRF-LysCTT, respectively, and were given starvation treatment (12 h) and H/R treatment (12 h) after 24 h of transfection. Primary cultured cardiomyocytes were divided into groups E, F, and G: the cardiomyocytes in group E were cultured in a complete medium for 24 h; those in group F were transfected with agomir-NC for 24 h; those in group G were transfected with agomir-5'tRF-LysCTT for 24 h. RT-qPCR was used to measure the relative expression levels of 5'tRF-LysCTT, Ptgs2, Gpx4, and Slc7a11 in mouse cardiomyocytes of groups A-G; ferrous ion colorimetric test kit, lipid reactive oxygen species (ROS) staining, and CCK8 assay kit were used to measure the relative content of ferrous ions, ROS level, and the survival rate of cardiomyocytes in groups A-G; Prussian blue staining was used to observe iron ion deposition in cardiomyocytes. Results RT-qPCR showed that the expression level of 5'tRF-LysCTT in heart was significantly higher than that in the liver, spleen, kidney, brain, and muscle (F=16.21,t=3.81-7.93,P0.05). Compared with group E, group G had significant increases in the relative expression levels of 5'tRF-LysCTT and Ptgs2, significant reductions in the relative expression levels of Gpx4 and Slc7a11, significant increases in the relative content of ferrous ions and ROS level, and a significant reduction in the survival rate of cardiomyocytes (t=3.57-91.84,P0.05). Conclusion 5'tRF-LysCTT can regulate ferroptosis in mouse cardiomyocytes, and knockdown of 5'tRF-LysCTT can inhibit cardiomyocyte ferroptosis induced by H/R, while overexpression of 5'tRF-LysCTT can promote cardiomyocyte ferroptosis.

Objective To investigate the regulatory effect of 3'-tRF-PheGAA on angiotensin Ⅱ (Ang Ⅱ)-induced cardiomyocyte hypertrophy and its mechanism. Methods Primary cardiomyocytes of neonatal mice were obtained and cultured for 24 h, and were then divided into groups A to G. The cardiomyocytes in group A were cultured with DMEM/F12 complete culture medium for 36 h. The cardiomyocytes in group B were transfected with negative control (NC) for 36 h. The cardiomyocytes in group C were transfected with agomir-3'-tRF-PheGAA for 36 h. The cardiomyocytes in group D were cultured with DMEM/F12 complete culture medium for 84 h. The cardiomyocytes in group E were treated with Ang Ⅱ at a final concentration of 1 μmol/L for 48 h. The cardiomyocytes in group F were transfected with anta-NC for 36 h and then treated with Ang Ⅱ at a final concentration of 1 μmol/L for 48 h. The cardiomyocytes in group G were transfected with anta-3'-tRF-PheGAA for 36 h and then treated with Ang Ⅱ at a final concentration of 1 μmol/L for 48 h. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to determine the gene expression levels of 3'-tRF-PheGAA, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β-myosin heavy chain (β-MHC) in the cardiomyocytes in each group. Rhodamine-labeled phalloidin was used to stain F-actin in the cardiomyocytes in each group and calculate the skeletal surface area of cardiomyocytes. Results The RT-qPCR results showed significant differences between groups A to C in the gene expression levels of 3'-tRF-PheGAA, ANP, BNP, and β-MHC in cardiomyocytes (F=137.10-1 061.00,P

BACKGROUND: Cardiomyocyte differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP (bone morphogenic protein)/Wnt-signaling pathways. However, the mechanisms and how regulatory roadblocks are removed with specific developmental signaling pathways remain unclear. METHODS: We conducted a genome-wide CRISPR screen to uncover essential regulators of cardiomyocyte specification in human embryonic stem cells using a myosin heavy chain 6 (MYH6)-GFP (green fluorescence protein) reporter system. After an independent secondary single guide ribonucleic acid validation of 25 candidates, we identified NF2 (neurofibromin 2), a moesin-ezrin-radixin like (MERLIN) tumor suppressor, as an upstream driver of early cardiomyocyte lineage specification. Independent monoclonal NF2 knockouts were generated using CRISPR-Cas9, and cell states were inferred through bulk RNA sequencing and protein expression analysis across differentiation time points. Terminal lineage differentiation was assessed by using an in vitro 2-dimensional-micropatterned gastruloid model, trilineage differentiation, and cardiomyocyte differentiation. Protein interaction and post-translation modification of NF2 with its interacting partners were assessed using site-directed mutagenesis, coimmunoprecipitation, and proximity ligation assays. RESULTS: Transcriptional regulation and trajectory inference from NF2 -null cells reveal the loss of cardiomyocyte identity and the acquisition of nonmesodermal identity. Sustained elevation of early mesoderm lineage repressor SOX2 and upregulation of late anticardiac regulators CDX2 and MSX1 in NF2 knockout cells reflect a necessary role for NF2 in removing regulatory roadblocks. Furthermore, we found that NF2 and AMOT (angiomotin) cooperatively bind to YAP (yes-associated protein) during mesendoderm formation, thereby preventing YAP activation, independent of canonical MST (mammalian sterile 20-like serine-threonine protein kinase)–LATS (large tumor suppressor serine-threonine protein kinase) signaling. Mechanistically, cardiomyocyte lineage identity was rescued by wild-type and NF2 serine-518 phosphomutants, but not NF2 FERM (ezrin-radixin-meosin homology protein) domain blue-box mutants, demonstrating that the critical FERM domain–dependent formation of the AMOT-NF2-YAP scaffold complex at the adherens junction is required for early cardiomyocyte lineage differentiation. CONCLUSIONS: These results provide mechanistic insight into the essential role of NF2 during early epithelial-mesenchymal transition by sequestering the repressive effect of YAP and relieving regulatory roadblocks en route to cardiomyocytes. [ABSTRACT FROM AUTHOR]

BACKGROUND: During the neonatal stage, the cardiomyocyte undergoes a constellation of molecular, cytoarchitectural, and functional changes known collectively as cardiomyocyte maturation to increase myocardial contractility and cardiac output. Despite the importance of cardiomyocyte maturation, the molecular mechanisms governing this critical process remain largely unexplored. METHODS: We leveraged an in vivo mosaic knockout system to characterize the role of Carm1 , the founding member of protein arginine methyltransferase, in cardiomyocyte maturation. Using a battery of assays, including immunohistochemistry, immuno-electron microscopy imaging, and action potential recording, we assessed the effect of loss of Carm1 function on cardiomyocyte cell growth, myofibril expansion, T-tubule formation, and electrophysiological maturation. Genome-wide transcriptome profiling, H3R17me2a chromatin immunoprecipitation followed by sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing were used to investigate the mechanisms by which CARM1 (coactivator-associated arginine methyltransferase 1) regulates cardiomyocyte maturation. Finally, we interrogated the human syntenic region to the H3R17me2a chromatin immunoprecipitation followed by sequencing peaks for single-nucleotide polymorphisms associated with human heart diseases. RESULTS: We report that mosaic ablation of Carm1 disrupts multiple aspects of cardiomyocyte maturation cell autonomously, leading to reduced cardiomyocyte size and sarcomere thickness, severe loss and disorganization of T tubules, and compromised electrophysiological maturation. Genomics study demonstrates that CARM1 directly activates genes that underlie cardiomyocyte cytoarchitectural and electrophysiological maturation. Moreover, our study reveals significant enrichment of human heart disease–associated single-nucleotide polymorphisms in the human genomic region syntenic to the H3R17me2a chromatin immunoprecipitation followed by sequencing peaks. CONCLUSIONS: This study establishes a critical and multifaceted role for CARM1 in regulating cardiomyocyte maturation and demonstrates that deregulation of CARM1-dependent cardiomyocyte maturation gene expression may contribute to human heart diseases. [ABSTRACT FROM AUTHOR]

This article discusses the use of nanopore sequencing to detect chemical modifications in mRNA, specifically N6-methyladenosine (m6A), which can affect gene regulation. The study focused on the role of the enzyme METTL3 in regulating cardiomyocyte mitochondrial metabolism. Through nanopore sequencing, the researchers identified 394 m6A-modified mRNAs, with the modifications most frequently found in the stop codon and 3' untranslated region. The study also found that the location of the m6A mark and differential binding of m6A recognition proteins can control the fate of the mRNA. The altered proteins in METTL3-deficient hearts were linked to mitochondrial function, suggesting that mitochondrial dysfunction may be a primary driver of cardiomyopathy. This study provides valuable insights into the role of mRNA modifications in cardiac homeostasis. [Extracted from the article]

BACKGROUND: TTN truncation variants (TTNtvs) are the most common genetic lesion identified in individuals with dilated cardiomyopathy, a disease with high morbidity and mortality rates. TTNtvs reduce normal TTN (titin) protein levels, produce truncated proteins, and impair sarcomere content and function. Therapeutics targeting TTNtvs have been elusive because of the immense size of TTN, the rarity of specific TTNtvs, and incomplete knowledge of TTNtv pathogenicity. METHODS: We adapted CRISPR activation using dCas9-VPR to functionally interrogate TTNtv pathogenicity and develop a therapeutic in human cardiomyocytes and 3-dimensional cardiac microtissues engineered from induced pluripotent stem cell models harboring a dilated cardiomyopathy–associated TTNtv. We performed guide RNA screening with custom TTN reporter assays, agarose gel electrophoresis to quantify TTN protein levels and isoforms, and RNA sequencing to identify molecular consequences of TTN activation. Cardiomyocyte epigenetic assays were also used to nominate DNA regulatory elements to enable cardiomyocyte-specific TTN activation. RESULTS: CRISPR activation of TTN using single guide RNAs targeting either the TTN promoter or regulatory elements in spatial proximity to the TTN promoter through 3-dimensional chromatin interactions rescued TTN protein deficits disturbed by TTNtvs. Increasing TTN protein levels normalized sarcomere content and contractile function despite increasing truncated TTN protein. In addition to TTN transcripts, CRISPR activation also increased levels of myofibril assembly-related and sarcomere-related transcripts. CONCLUSIONS: TTN CRISPR activation rescued TTNtv-related functional deficits despite increasing truncated TTN levels, which provides evidence to support haploinsufficiency as a relevant genetic mechanism underlying heterozygous TTNtvs. CRISPR activation could be developed as a therapeutic to treat a large proportion of TTNtvs. [ABSTRACT FROM AUTHOR]

This article explores the pathogenesis of ventricular hypoplasia in pulmonary atresia with intact ventricular septum (PA-IVS), a type of hypoplastic right heart syndrome. The study utilizes patient-specific induced pluripotent stem cells (iPSCs) and single-cell RNA sequencing to investigate the differentiation and proliferation of cardiomyocytes in PA-IVS. The findings suggest that impaired cardiomyocyte proliferation and abnormal lineage differentiation may contribute to the development of right ventricle hypoplasia in PA-IVS. Additionally, metabolic dysfunction and elevated mitochondrial activity are observed in PA-IVS iPSC-derived cardiomyocytes. Further research using 3-dimensional iPSC-derived cardiac organoids is recommended to better understand the specific defects in hypoplastic right heart syndrome. [Extracted from the article]

This document, published in the journal Circulation, discusses the identification of cardiomyocyte ploidy using nuclear area and volume. The authors developed a method to estimate the nuclear ploidy of cardiomyocytes that is accurate and accessible for cardiac research. They used fluorescence in situ hybridization and confocal microscopy to analyze the nuclear areas and volumes of cardiomyocytes. The results showed that nuclear volume is a reliable metric for assessing cardiomyocyte ploidy, both in isolated cells and tissue sections. This method has potential applications in pathology and disease research. [Extracted from the article]

BACKGROUND: Proper nuclear organization is critical for cardiomyocyte function, because global structural remodeling of nuclear morphology and chromatin structure underpins the development and progression of cardiovascular disease. Previous reports have implicated a role for DNA damage in cardiac hypertrophy; however, the mechanism for this process is not well delineated. AMPK (AMP-activated protein kinase) family of proteins regulates metabolism and DNA damage response (DDR). Here, we examine whether a member of this family, SNRK (SNF1-related kinase), which plays a role in cardiac metabolism, is also involved in hypertrophic remodeling through changes in DDR and structural properties of the nucleus. METHODS: We subjected cardiac-specific Snrk-/- mice to transaortic banding to assess the effect on cardiac function and DDR. In parallel, we modulated SNRK in vitro and assessed its effects on DDR and nuclear parameters. We also used phosphoproteomics to identify novel proteins that are phosphorylated by SNRK. Last, coimmunoprecipitation was used to verify Destrin (DSTN) as the binding partner of SNRK that modulates its effects on the nucleus and DDR. RESULTS: Cardiac-specific Snrk-/- mice display worse cardiac function and cardiac hypertrophy in response to transaortic banding, and an increase in DDR marker pH2AX (phospho-histone 2AX) in their hearts. In addition, in vitro Snrk knockdown results in increased DNA damage and chromatin compaction, along with alterations in nuclear flatness and 3-dimensional volume. Phosphoproteomic studies identified a novel SNRK target, DSTN, a member of F-actin depolymerizing factor proteins that directly bind to and depolymerize F-actin. SNRK binds to DSTN, and DSTN downregulation reverses excess DNA damage and changes in nuclear parameters, in addition to cellular hypertrophy, with SNRK knockdown. We also demonstrate that SNRK knockdown promotes excessive actin depolymerization, measured by the increased ratio of G-actin to F-actin. Last, jasplakinolide, a pharmacological stabilizer of F-actin, rescues the increased DNA damage and aberrant nuclear morphology in SNRK-downregulated cells. CONCLUSIONS: These results indicate that SNRK is a key player in cardiac hypertrophy and DNA damage through its interaction with DSTN. This interaction fine-tunes actin polymerization to reduce DDR and maintain proper cardiomyocyte nuclear shape and morphology. [ABSTRACT FROM AUTHOR]

Background: Shortly after birth, cardiomyocytes exit the cell cycle and cease proliferation. At present, the regulatory mechanisms for this loss of proliferative capacity are poorly understood. CBX7 (chromobox 7), a polycomb group (PcG) protein, regulates the cell cycle, but its role in cardiomyocyte proliferation is unknown. Methods: We profiled CBX7 expression in the mouse hearts through quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry. We overexpressed CBX7 in neonatal mouse cardiomyocytes through adenoviral transduction. We knocked down CBX7 by using constitutive and inducible conditional knockout mice (Tnnt2-Cre;Cbx7fl/+ and Myh6-MCM;Cbx7fl/fl , respectively). We measured cardiomyocyte proliferation by immunostaining of proliferation markers such as Ki67, phospho-histone 3, and cyclin B1. To examine the role of CBX7 in cardiac regeneration, we used neonatal cardiac apical resection and adult myocardial infarction models. We examined the mechanism of CBX7-mediated repression of cardiomyocyte proliferation through coimmunoprecipitation, mass spectrometry, and other molecular techniques. Results: We explored Cbx7 expression in the heart and found that mRNA expression abruptly increased after birth and was sustained throughout adulthood. Overexpression of CBX7 through adenoviral transduction reduced proliferation of neonatal cardiomyocytes and promoted their multinucleation. On the other hand, genetic inactivation of Cbx7 increased proliferation of cardiomyocytes and impeded cardiac maturation during postnatal heart growth. Genetic ablation of Cbx7 promoted regeneration of neonatal and adult injured hearts. Mechanistically, CBX7 interacted with TARDBP (TAR DNA-binding protein 43) and positively regulated its downstream target, RBM38 (RNA Binding Motif Protein 38), in a TARDBP-dependent manner. Overexpression of RBM38 inhibited the proliferation of CBX7-depleted neonatal cardiomyocytes. Conclusions: Our results demonstrate that CBX7 directs the cell cycle exit of cardiomyocytes during the postnatal period by regulating its downstream targets TARDBP and RBM38. This is the first study to demonstrate the role of CBX7 in regulation of cardiomyocyte proliferation, and CBX7 could be an important target for cardiac regeneration. [ABSTRACT FROM AUTHOR]

Objective To study the protective effect of Shexiang Baoxin Pill (SBP) on hypoxia-reoxygenation cardiomyocytes and its effect on apoptosis. Methods The model of hypoxia and reoxygenation of cardiomyocytes was established. The active components of SBP were extracted by water extraction. According to whether different concentrations of SBP active components were given, cells were divided into the control group, the model group, the 0.5 g/L SBP group, the 1 g/L SBP group, the 2 g/L SBP group and the 5 g/L SBP group. The highest survival rate in the SBP intervention group was selected as the group for the follow-up experiments. The cell survival rate of each group was detected by CCK-8. The apoptosis rate of each group was detected by Anexxin-VFITC/PI double staining. Western bolt assay was used to detect the relative expression levels of B cell lymphoma factor 2 (Bcl-2), Bcl-2 associated X protein (Bax) and cysteine protease 3 (Caspase-3) in each group. Results Compared with the model group, survival rates of cardiomyocytes were increased in the 0.5 g/L, 1 g/L, 2 g/L and 5 g/L SBP groups (P＜0.01), and the survival rate of cardiomyocytes was the highest in the 2 g/L SBP group. Compared with the control group, the apoptosis rate, relative expression levels of Bax and Caspase-3 were increased in the model group and the 2 g/L SBP group (P＜0.05), but there was no significant difference in Bcl-2 between the groups. Compared with the model group, the apoptosis rate, relative expression levels of Bax and Caspase-3 were decreased in the 2 g/L SBP group (P＜0.05), while relative expression levels of Bcl-2 and Bcl-2/Bax ratio were increased (P＜0.05). Conclusion Shexiang Baoxin Pill has a protective effect on hypoxia-reoxygenation cardiomyocytes, and the inhibition of apoptosis is one of its protective mechanisms. [ABSTRACT FROM AUTHOR]

Background: During cardiomyocyte maturation, the centrosome, which functions as a microtubule organizing center in cardiomyocytes, undergoes dramatic structural reorganization where its components reorganize from being localized at the centriole to the nuclear envelope. This developmentally programmed process, referred to as centrosome reduction, has been previously associated with cell cycle exit. However, understanding of how this process influences cardiomyocyte cell biology, and whether its disruption results in human cardiac disease, remains unknown. We studied this phenomenon in an infant with a rare case of infantile dilated cardiomyopathy (iDCM) who presented with left ventricular ejection fraction of 18% and disrupted sarcomere and mitochondria structure. Methods: We performed an analysis beginning with an infant who presented with a rare case of iDCM. We derived induced pluripotent stem cells from the patient to model iDCM in vitro. We performed whole exome sequencing on the patient and his parents for causal gene analysis. CRISPR/Cas9-mediated gene knockout and correction in vitro were used to confirm whole exome sequencing results. Zebrafish and Drosophila models were used for in vivo validation of the causal gene. Matrigel mattress technology and single-cell RNA sequencing were used to characterize iDCM cardiomyocytes further. Results: Whole exome sequencing and CRISPR/Cas9 gene knockout/correction identified RTTN , the gene encoding the centrosomal protein RTTN (rotatin), as the causal gene underlying the patient's condition, representing the first time a centrosome defect has been implicated in a nonsyndromic dilated cardiomyopathy. Genetic knockdowns in zebrafish and Drosophila confirmed an evolutionarily conserved requirement of RTTN for cardiac structure and function. Single-cell RNA sequencing of iDCM cardiomyocytes showed impaired maturation of iDCM cardiomyocytes, which underlie the observed cardiomyocyte structural and functional deficits. We also observed persistent localization of the centrosome at the centriole, contrasting with expected programmed perinuclear reorganization, which led to subsequent global microtubule network defects. In addition, we identified a small molecule that restored centrosome reorganization and improved the structure and contractility of iDCM cardiomyocytes. Conclusions: This study is the first to demonstrate a case of human disease caused by a defect in centrosome reduction. We also uncovered a novel role for RTTN in perinatal cardiac development and identified a potential therapeutic strategy for centrosome-related iDCM. Future study aimed at identifying variants in centrosome components may uncover additional contributors to human cardiac disease. [ABSTRACT FROM AUTHOR]

Objective To explore the mechanism of transcription factor EB (TFEB) in autophagy of aging cardiomyocytes. Methods Animal experiment: Twenty aged Wistar rats were randomly divided into the sham operation group (Sham group) and the ischemia-reperfusion injury group (I/R group). Cell experiments: (1) Aging cardiomyocytes were cultured in vitro, incubated with 8 g/L D-galactose for 8 days, and then divided into the normoxia group and the hypoxia/ reoxygenation group (H/R group). (2) Aging cardiomyocytes transfected by adenovirus overexpressing and interfering with TFEB were divided into the Ad-GFP group, the Ad-GFP+H/R group, the Ad-TFEB group, the Ad-TFEB+H/R group, the sh-NC group, the sh-NC+H/R group, the sh-TFEB group and the sh-TFEB+H/R group. (3) Aging cardiomyocytes treated with specific inhibitors of DNMT1, DNMT3a and DNMT3b after hypoxia/reoxygenation were divided into the H/R group, the DNMT1 specific inhibitor (DC-05) group, the DNMT3a specific inhibitor (TFD) group and the DNMT3b specific inhibitor (NA) group. (4) Aging cardiomyocytes transfected by adenovirus interfering with DNMT3b were divided into the sh-NC group, the sh-NC+H/R group, the sh-DNMT3b group and the sh-DNMT3b+H/R group. Quantitative real-time PCR (qPCR) was used to detect the mRNA level of TFEB, and Western blot assay was used to detect the autophagy related proteins TFEB, LC3B and p62 in aging cardiomyocytes. The DNA methylation levels of TFEB promoter in aging myocardium and cardiomyocytes were detected by nested methylation specific PCR (nMS-PCR). Results Compared with the Sham group or the normoxia group, the mRNA and protein expression of TFEB were decreased in the I/R group and the H/R group (P＜ 0.01). The protein expression of LC3B-Ⅱ/Ⅰ was decreased in aging cardiomyocytes after overexpression of TFEB in the Ad-TFEB group compared with the Ad-GFP group, while the protein expression of p62 was increased (P＜0.01). The opposite results were obtained after interfering with TFEB (P＜0.01). Compared with the Sham group or the normoxia group, the DNA methylation level of the TFEB promoter was increased in the I/R group and the H/R group (P＜0.05). Compared with the H/R group, it was found that the mRNA and protein expression level of TFEB were increased in the NA group (P＜ 0.01). And the TFEB mRNA and protein expression were increased in aging cardiomyocytes after overexpressed interference with DNMT3b (P＜0.01). Conclusion DNMT3b inhibits the TFEB expression by regulating DNA methylation of TFEB promoter, thus to promote autophagy of aging cardiomyocytes. [ABSTRACT FROM AUTHOR]

Background: Developmental cardiac tissue holds remarkable capacity to regenerate after injury and consists of regenerative mononuclear and diploid cardiomyocytes (MNDCMs). Upon maturation, MNDCMs become binucleated or polyploid and exit the cell cycle. Interestingly, cardiomyocyte (CM) metabolism undergoes a profound shift that coincides with cessation of regeneration in the postnatal heart. However, whether reprogramming metabolism promotes persistence of regenerative MNDCMs enhancing cardiac function and repair after injury is unknown. Here, we identify a novel role for RNA-binding protein LIN28a, a master regulator of cellular metabolism, in cardiac repair following injury. Methods: LIN28a overexpression was tested using mouse transgenesis on postnatal CM numbers, cell cycle and response to apical resection (AR) injury. Using, neonatal and adult cell culture system and adult and MADM myocardial injury models in mice, effect of LIN28a overexpression on cardiomyocyte cell cycle and metabolism was tested. Finally, isolated adult CMs from LIN28a and wildtype mice 4 days after myocardial injury, were used for RNA-immunoprecipitation sequencing (RIP-seq). Results: LIN28a was found as primarily active during cardiac development and rapidly decreases after birth. LIN28a reintroduction at P1, P3, P5, and P7 decreased maturation-associated polyploidization, nucleation, and cell size, enhancing CM cell cycle activity in LIN28a transgenic pups compared to WT littermates. Moreover, LIN28a overexpression extended CM cell cycle activity beyond P7 concurrent with increased cardiac function 30 days after AR. In the adult heart, LIN28a overexpression attenuated CM apoptosis, enhanced cell cycle activity, cardiac function, and survival in mice 12 weeks after myocardial infarction compared to WT littermate controls. Alternatively, LIN28a small molecule inhibitor attenuated pro-reparative effects of LIN28a on the heart. Mechanistically, Neonatal rat ventricular myocytes (NRVMs) overexpressing LIN28a showed increased glycolysis, ATP production and levels of metabolic enzymes compared to control. LIN28a immunoprecipitation followed by RNA sequencing (RIPseq) in CMs isolated from LIN28a-overexpressing hearts after injury identified lncRNA-H19 as its most significantly altered target. Ablation of lncRNA-H19 blunted LIN28a-induced enhancement on CM metabolism and cell cycle activity. Conclusions: Collectively, LIN28a reprograms CM metabolism and promotes persistence of MNDCMs in the injured heart enhancing pro-reparative processes thereby linking CM metabolism to regulation of ploidy/nucleation and repair in the heart. [ABSTRACT FROM AUTHOR]

Background: Because adult cardiomyocytes have little regenerative capacity, resident cardiac fibroblasts (CFs) synthesize extracellular matrix after myocardial infarction (MI) to form fibrosis, leading to cardiac dysfunction and heart failure. Therapies that can regenerate the myocardium and reverse fibrosis in chronic MI are lacking. The overexpression of cardiac transcription factors, including Mef2c/Gata4/Tbx5/Hand2 (MGTH), can directly reprogram CFs into induced cardiomyocytes (iCMs) and improve cardiac function under acute MI. However, the ability of in vivo cardiac reprogramming to repair chronic MI with established scars is undetermined. Methods: We generated a novel Tcf21iCre/reporter/MGTH2A transgenic mouse system in which tamoxifen treatment could induce both MGTH and reporter expression in the resident CFs for cardiac reprogramming and fibroblast lineage tracing. We first tested the efficacy of this transgenic system in vitro and in vivo for acute MI. Next, we analyzed in vivo cardiac reprogramming and fusion events under chronic MI using Tcf21iCre/Tomato/MGTH2A and Tcf21iCre/mTmG/MGTH2A mice, respectively. Microarray and single-cell RNA sequencing were performed to determine the mechanism of cardiac repair by in vivo reprogramming. Results: We confirmed the efficacy of transgenic in vitro and in vivo cardiac reprogramming for acute MI. In chronic MI, in vivo cardiac reprogramming converted ≈2% of resident CFs into iCMs, in which a majority of iCMs were generated by means of bona fide cardiac reprogramming rather than by fusion with cardiomyocytes. Cardiac reprogramming significantly improved myocardial contraction and reduced fibrosis in chronic MI. Microarray analyses revealed that the overexpression of MGTH activated cardiac program and concomitantly suppressed fibroblast and inflammatory signatures in chronic MI. Single-cell RNA sequencing demonstrated that resident CFs consisted of 7 subclusters, in which the profibrotic CF population increased under chronic MI. Cardiac reprogramming suppressed fibroblastic gene expression in chronic MI by means of conversion of profibrotic CFs to a quiescent antifibrotic state. MGTH overexpression induced antifibrotic effects partly by suppression of Meox1, a central regulator of fibroblast activation. Conclusions: These results demonstrate that cardiac reprogramming could repair chronic MI by means of myocardial regeneration and reduction of fibrosis. These findings present opportunities for the development of new therapies for chronic MI and heart failure. [ABSTRACT FROM AUTHOR]

Background: Identifying genetic variants which impact the level of cell cycle reentry and establishing the degree of cell cycle progression in those variants could help guide development of therapeutic interventions aimed at effecting cardiac regeneration. We observed that C57Bl6/NCR (B6N) mice have a marked increase in cardiomyocyte S-phase activity following permanent coronary artery ligation as compared to infarcted DBA/2J (D2J) mice.Methods: Cardiomyocyte cell cycle activity post-infarction was monitored in D2J, (D2J x B6N)-F1 and [(D2J x B6N)-F1 x D2J] backcross mice via bromodeoxyuridine or 5-ethynyl-2' -deoxyuridine incorporation, using a nuclear-localized transgenic reporter to identify cardiomyocyte nuclei. Genome-wide quantitative trait locus (QTL) analysis, fine scale genetic mapping, whole exome sequencing and RNA-seq analyses of the backcross mice were performed to identify the gene responsible for the elevated cardiomyocyte S-phase phenotype.Results: (D2J x B6N)-F1 mice exhibited a 14-fold increase in cardiomyocyte S-phase activity in ventricular regions remote from infarct scar as compared to D2J mice (0.798 ± 0.09% vs. 0.056 ± 0.004%; p < 0.001). QTL analysis of [(D2J x B6N)-F1 x D2J] backcross mice revealed that the gene responsible for differential S-phase activity was located on the distal arm of Chromosome 3 (LOD score = 6.38; p < 0.001). Additional genetic and molecular analyses identified 3 potential candidates. Of these, troponin I-interacting kinase (Tnni3k) is expressed in B6N hearts but not in D2J hearts. Transgenic expression of Tnni3k in a D2J genetic background results in elevated cardiomyocyte S-phase activity post-injury. Cardiomyocyte S-phase activity in both TNNI3K-expressing and TNNI3K-nonexpressing mice results in the formation of polyploid nuclei.Conclusions: These data indicate that TNNI3K expression increases the level of cardiomyocyte S-phase activity following injury. [ABSTRACT FROM AUTHOR]

Background: Transcatheter aortic valve replacement (TAVR) is a well-established treatment option for high- and intermediate-risk patients with severe symptomatic aortic valve stenosis. A majority of patients exhibit improvements in left ventricular ejection fraction (LVEF) after TAVR in response to TAVR-associated afterload reduction. However, a specific role for circulating microRNAs (miRNAs) in the improvement of cardiac function for patients after TAVR has not yet been investigated. Here, we profiled the differential expression of miRNAs in circulating extracellular vesicles (EVs) in patients after TAVR and, in particular, the novel role of circulating miR-122-5p in cardiomyocytes. Methods: Circulating EV-associated miRNAs were investigated by use of an unbiased Taqman-based human miRNA array. Several EV miRNAs (miR-122-5p, miR-26a, miR-192, miR-483-5p, miR-720, miR-885-5p, and miR-1274) were significantly deregulated in patients with aortic valve stenosis at day 7 after TAVR compared with the preprocedural levels in patients without LVEF improvement. The higher levels of miR-122-5p were negatively correlated with LVEF improvement at both day 7 (r =−0.264 and P =0.015) and 6 months (r =−0.328 and P =0.0018) after TAVR. Results: Using of patient-derived samples and a murine aortic valve stenosis model, we observed that the expression of miR-122-5p correlates negatively with cardiac function, which is associated with LVEF. Mice with graded wire injury–induced aortic valve stenosis demonstrated a higher level of miR-122-5p, which was related to cardiomyocyte dysfunction. Murine ex vivo experiments revealed that miR-122-5p is highly enriched in endothelial cells compared with cardiomyocytes. Coculture experiments, copy-number analysis, and fluorescence microscopy with Cy3-labeled miR-122-5p demonstrated that miR-122-5p can be shuttled through large EVs from endothelial cells into cardiomyocytes. Gain- and loss-of-function experiments suggested that EV-mediated shuttling of miR-122-5p increases the level of miR-122-5p in recipient cardiomyocytes. Mechanistically, mass spectrometry, miRNA pulldown, electrophoretic mobility shift assay, and RNA immunoprecipitation experiments confirmed that miR-122-5p interacts with the RNA-binding protein hnRNPU (heterogeneous nuclear ribonucleoprotein U) in a sequence-specific manner to encapsulate miR-122-5p into large EVs. On shuttling, miR-122-5p reduces the expression of the antiapoptotic gene BCL2 by binding to its 3′ untranslated region to inhibit its translation, thereby decreasing the viability of target cardiomyocytes. Conclusions: Increased levels of circulating proapoptotic EV-incorporated miR-122-5p are associated with reduced LVEF after TAVR. EV shuttling of miR-122-5p regulates the viability and apoptosis of cardiomyocytes in a BCL2-dependent manner. [ABSTRACT FROM AUTHOR]

Background: Direct cardiac reprogramming of fibroblasts into cardiomyocytes has emerged as a promising strategy to remuscularize injured myocardium. However, it is insufficient to generate functional induced cardiomyocytes from human fibroblasts using conventional reprogramming cocktails, and the underlying molecular mechanisms are not well studied.Methods: To discover potential missing factors for human direct reprogramming, we performed transcriptomic comparison between human induced cardiomyocytes and functional cardiomyocytes.Results: We identified TBX20 (T-box transcription factor 20) as the top cardiac gene that is unable to be activated by the MGT133 reprogramming cocktail (MEF2C, GATA4, TBX5, and miR-133). TBX20 is required for normal heart development and cardiac function in adult cardiomyocytes, yet its role in cardiac reprogramming remains undefined. We show that the addition of TBX20 to the MGT133 cocktail (MGT+TBX20) promotes cardiac reprogramming and activates genes associated with cardiac contractility, maturation, and ventricular heart. Human induced cardiomyocytes produced with MGT+TBX20 demonstrated more frequent beating, calcium oscillation, and higher energy metabolism as evidenced by increased mitochondria numbers and mitochondrial respiration. Mechanistically, comprehensive transcriptomic, chromatin occupancy, and epigenomic studies revealed that TBX20 colocalizes with MGT reprogramming factors at cardiac gene enhancers associated with heart contraction, promotes chromatin binding and co-occupancy of MGT factors at these loci, and synergizes with MGT for more robust activation of target gene transcription.Conclusions: TBX20 consolidates MGT cardiac reprogramming factors to activate cardiac enhancers to promote cardiac cell fate conversion. Human induced cardiomyocytes generated with TBX20 showed enhanced cardiac function in contractility and mitochondrial respiration. [ABSTRACT FROM AUTHOR]

Background: Cardiac hypertrophy is initially an adaptive response of cardiomyocytes to neurohumoral or hemodynamic stimuli. Evidence indicates that Ang II (angiotensin II) or pressure overload causes GSDMD (gasdermin D) activation in cardiomyocytes and myocardial tissues. However, the direct impact of GSDMD on cardiac hypertrophy and its underlying mechanisms are not fully understood.Methods and Results: In this study, we examined the aberrant activation of GSDMD in mouse and human hypertrophic myocardia, and the results showed that GSDMD deficiency reduced Ang II or pressure overload-induced cardiac hypertrophy, dysfunction, and associated cardiomyocyte pyroptosis in mice. Mechanistically, Ang II-mediated GSDMD cleavage caused mitochondrial dysfunction upstream of STING (stimulator of interferon genes) activation in vivo and in vitro. Activation of STING, in turn, potentiated GSDMD-mediated cardiac hypertrophy. Moreover, deficiency of both GSDMD and STING suppressed cardiac hypertrophy in cardiac-specific GSDMD-overexpressing mice.Conclusions: Based on these findings, we propose a mechanism by which GSDMD generates a self-amplifying, positive feed-forward loop with the mitochondria-STING axis. This finding points to the prospects of GSDMD as a key therapeutic target for hypertrophy-associated heart diseases. [ABSTRACT FROM AUTHOR]

Background: Cell-free DNA (cfDNA) is a noninvasive marker of cellular injury. Its significance in pulmonary arterial hypertension (PAH) is unknown.Methods: Plasma cfDNA was measured in 2 PAH cohorts (A, n=48; B, n=161) and controls (n=48). Data were collected for REVEAL 2.0 (Registry to Evaluate Early and Long-Term PAH Disease Management) scores and outcome determinations. Patients were divided into the following REVEAL risk groups: low (≤6), medium (7-8), and high (≥9). Total cfDNA concentrations were compared among controls and PAH risk groups by 1-way analysis of variance. Log-rank tests compared survival between cfDNA tertiles and REVEAL risk groups. Areas under the receiver operating characteristic curve were estimated from logistic regression models. A sample subset from cohort B (n=96) and controls (n=16) underwent bisulfite sequencing followed by a deconvolution algorithm to map cell-specific cfDNA methylation patterns, with concentrations compared using t tests.Results: In cohort A, median (interquartile range) age was 62 years (47-71), with 75% female, and median (interquartile range) REVEAL 2.0 was 6 (4-9). In cohort B, median (interquartile range) age was 59 years (49-71), with 69% female, and median (interquartile range) REVEAL 2.0 was 7 (6-9). In both cohorts, cfDNA concentrations differed among patients with PAH of varying REVEAL risk and controls (analysis of variance P≤0.002) and were greater in the high-risk compared with the low-risk category (P≤0.002). In cohort B, death or lung transplant occurred in 14 of 54, 23 of 53, and 35 of 54 patients in the lowest, middle, and highest cfDNA tertiles, respectively. cfDNA levels stratified as tertiles (log-rank: P=0.0001) and REVEAL risk groups (log-rank: P<0.0001) each predicted transplant-free survival. The addition of cfDNA to REVEAL improved discrimination (area under the receiver operating characteristic curve, 0.72-0.78; P=0.02). Compared with controls, methylation analysis in patients with PAH revealed increased cfDNA originating from erythrocyte progenitors, neutrophils, monocytes, adipocytes, natural killer cells, vascular endothelium, and cardiac myocytes (Bonferroni adjusted P<0.05). cfDNA concentrations derived from erythrocyte progenitor cells, cardiac myocytes, and vascular endothelium were greater in patients with PAH with high-risk versus low-risk REVEAL scores (P≤0.02).Conclusions: Circulating cfDNA is elevated in patients with PAH, correlates with disease severity, and predicts worse survival. Results from cfDNA methylation analyses in patients with PAH are consistent with prevailing paradigms of disease pathogenesis. [ABSTRACT FROM AUTHOR]

ABSTRACT Objective Analyze the effects of aerobic and anaerobic training on different ergometers on muscle and cardiac hypertrophy in rats. Methods The animals were separated into the following groups: Control (C), Aerobic Training in Water (ATW), Resistance Training in Water (RTW), Aerobic Training on Treadmill (ATT), and Resistance Training in Climbing (RTC). All training protocols were carried out for 4 weeks, 3 times/week. The cross-sectional area (CSA) of the gastrocnemius muscle cells and the areas of the cardiomyocytes were measured. Results In the fast-twitch fibers, there was an increase in CSA in the RTW and RTC groups compared to the ATW (p

The hypothesis that telomeres fuse to generate polyploid cardiomyocytes predicts that telomere fusion will be less frequent in diploid than in polyploid cardiomyocytes. Keywords: myocytes, cardiac; polyploidy; telomere EN myocytes, cardiac polyploidy telomere 1634 1636 3 05/22/23 20230523 NES 230523 Although polyploidization is a hallmark of adult mammalian cardiomyocytes and may constrain their proliferation, the mechanisms leading to ploidy increase in cardiomyocytes remain elusive.[1] Our laboratory and others have reported the formation of DNA bridges between daughter nuclei as a potential route to cardiomyocyte polyploidization.[2],[3] These earlier in vivo studies found DNA bridges on thin tissue sections, but this approach does not cover all events because not all cardiomyocytes were oriented in the same 2-dimensional plane. [Extracted from the article]

Background: Cytokines such as tumor necrosis factor-α (TNFα) have been implicated in cardiac dysfunction and toxicity associated with doxorubicin (DOX). Although TNFα can elicit different cellular responses, including survival or death, the mechanisms underlying these divergent outcomes in the heart remain cryptic. The E3 ubiquitin ligase TRAF2 (TNF receptor associated factor 2) provides a critical signaling platform for K63-linked polyubiquitination of RIPK1 (receptor interacting protein 1), crucial for nuclear factor-κB (NF-κB) activation by TNFα and survival. Here, we investigate alterations in TNFα-TRAF2-NF-κB signaling in the pathogenesis of DOX cardiotoxicity.Methods: Using a combination of in vivo (4 weekly injections of DOX 5 mg·kg-1·wk-1) in C57/BL6J mice and in vitro approaches (rat, mouse, and human inducible pluripotent stem cell-derived cardiac myocytes), we monitored TNFα levels, lactate dehydrogenase, cardiac ultrastructure and function, mitochondrial bioenergetics, and cardiac cell viability.Results: In contrast to vehicle-treated mice, ultrastructural defects, including cytoplasmic swelling, mitochondrial perturbations, and elevated TNFα levels, were observed in the hearts of mice treated with DOX. While investigating the involvement of TNFα in DOX cardiotoxicity, we discovered that NF-κB was readily activated by TNFα. However, TNFα-mediated NF-κB activation was impaired in cardiac myocytes treated with DOX. This coincided with loss of K63- linked polyubiquitination of RIPK1 from the proteasomal degradation of TRAF2. Furthermore, TRAF2 protein abundance was markedly reduced in hearts of patients with cancer treated with DOX. We further established that the reciprocal actions of the ubiquitinating and deubiquitinating enzymes cellular inhibitors of apoptosis 1 and USP19 (ubiquitin-specific peptidase 19), respectively, regulated the proteasomal degradation of TRAF2 in DOX-treated cardiac myocytes. An E3-ligase mutant of cellular inhibitors of apoptosis 1 (H588A) or gain of function of USP19 prevented proteasomal degradation of TRAF2 and DOX-induced cell death. Furthermore, wild-type TRAF2, but not a RING finger mutant defective for K63-linked polyubiquitination of RIPK1, restored NF-κB signaling and suppressed DOX-induced cardiac cell death. Last, cardiomyocyte-restricted expression of TRAF2 (cardiac troponin T-adeno-associated virus 9-TRAF2) in vivo protected against mitochondrial defects and cardiac dysfunction induced by DOX.Conclusions: Our findings reveal a novel signaling axis that functionally connects the cardiotoxic effects of DOX to proteasomal degradation of TRAF2. Disruption of the critical TRAF2 survival pathway by DOX sensitizes cardiac myocytes to TNFα-mediated necrotic cell death and DOX cardiotoxicity. [ABSTRACT FROM AUTHOR]

Background: GATA4 (GATA-binding protein 4), a zinc finger-containing, DNA-binding transcription factor, is essential for normal cardiac development and homeostasis in mice and humans, and mutations in this gene have been reported in human heart defects. Defects in alternative splicing are associated with many heart diseases, yet relatively little is known about how cell type- or cell state-specific alternative splicing is achieved in the heart. Here, we show that GATA4 regulates cell type-specific splicing through direct interaction with RNA and the spliceosome in human induced pluripotent stem cell-derived cardiac progenitors.Methods: We leveraged a combination of unbiased approaches including affinity purification of GATA4 and mass spectrometry, enhanced cross-linking with immunoprecipitation, electrophoretic mobility shift assays, in vitro splicing assays, and unbiased transcriptomic analysis to uncover GATA4's novel function as a splicing regulator in human induced pluripotent stem cell-derived cardiac progenitors.Results: We found that GATA4 interacts with many members of the spliceosome complex in human induced pluripotent stem cell-derived cardiac progenitors. Enhanced cross-linking with immunoprecipitation demonstrated that GATA4 also directly binds to a large number of mRNAs through defined RNA motifs in a sequence-specific manner. In vitro splicing assays indicated that GATA4 regulates alternative splicing through direct RNA binding, resulting in functionally distinct protein products. Correspondingly, knockdown of GATA4 in human induced pluripotent stem cell-derived cardiac progenitors resulted in differential alternative splicing of genes involved in cytoskeleton organization and calcium ion import, with functional consequences associated with the protein isoforms.Conclusions: This study shows that in addition to its well described transcriptional function, GATA4 interacts with members of the spliceosome complex and regulates cell type-specific alternative splicing via sequence-specific interactions with RNA. Several genes that have splicing regulated by GATA4 have functional consequences and many are associated with dilated cardiomyopathy, suggesting a novel role for GATA4 in achieving the necessary cardiac proteome in normal and stress-responsive conditions. [ABSTRACT FROM AUTHOR]

Background: Abnormalities in Ca2+ homeostasis are associated with cardiac arrhythmias and heart failure. Triadin plays an important role in Ca2+ homeostasis in cardiomyocytes. Alternative splicing of a single triadin gene produces multiple triadin isoforms. The cardiac-predominant isoform, mouse MT-1 or human Trisk32, is encoded by triadin exons 1 to 8. In humans, mutations in the triadin gene that lead to a reduction in Trisk32 levels in the heart can cause cardiac dysfunction and arrhythmias. Decreased levels of Trisk32 in the heart are also common in patients with heart failure. However, mechanisms that maintain triadin isoform composition in the heart remain elusive.Methods: We analyzed triadin expression in heart explants from patients with heart failure and cardiac arrhythmias and in hearts from mice carrying a knockout allele for Trdn-as, a cardiomyocyte-specific long noncoding RNA encoded by the antisense strand of the triadin gene, between exons 9 and 11. Catecholamine challenge with isoproterenol was performed on Trdn-as knockout mice to assess the role of Trdn-as in cardiac arrhythmogenesis, as assessed by ECG. Ca2+ transients in adult mouse cardiomyocytes were measured with the IonOptix platform or the GCaMP system. Biochemistry assays, single-molecule fluorescence in situ hybridization, subcellular localization imaging, RNA sequencing, and molecular rescue assays were used to investigate the mechanisms by which Trdn-as regulates cardiac function and triadin levels in the heart.Results: We report that Trdn-as maintains cardiac function, at least in part, by regulating alternative splicing of the triadin gene. Knockout of Trdn-as in mice downregulates cardiac triadin, impairs Ca2+ handling, and causes premature death. Trdn-as knockout mice are susceptible to cardiac arrhythmias in response to catecholamine challenge. Normalization of cardiac triadin levels in Trdn-as knockout cardiomyocytes is sufficient to restore Ca2+ handling. Last, Trdn-as colocalizes and interacts with serine/arginine splicing factors in cardiomyocyte nuclei and is essential for efficient recruitment of splicing factors to triadin precursor mRNA.Conclusions: These findings reveal regulation of alternative splicing as a novel mechanism by which a long noncoding RNA controls cardiac function. This study indicates potential therapeutics for heart disease by targeting the long noncoding RNA or pathways regulating alternative splicing. [ABSTRACT FROM AUTHOR]

Background: Certain nonmammalian species such as zebrafish have an elevated capacity for innate heart regeneration. Understanding how heart regeneration occurs in these contexts can help illuminate cellular and molecular events that can be targets for heart failure prevention or treatment. The epicardium, a mesothelial tissue layer that encompasses the heart, is a dynamic structure that is essential for cardiac regeneration in zebrafish. The extent to which different cell subpopulations or states facilitate heart regeneration requires research attention.Methods: To dissect epicardial cell states and associated proregenerative functions, we performed single-cell RNA sequencing and identified 7 epicardial cell clusters in adult zebrafish, 3 of which displayed enhanced cell numbers during regeneration. We identified paralogs of hapln1 as factors associated with the extracellular matrix and largely expressed in cluster 1. We assessed HAPLN1 expression in published single-cell RNA sequencing data sets from different stages and injury states of murine and human hearts, and we performed molecular genetics to determine the requirements for hapln1-expressing cells and functions of each hapln1 paralog.Results: A particular cluster of epicardial cells had the strongest association with regeneration and was marked by expression of hapln1a and hapln1b. The hapln1 paralogs are expressed in epicardial cells that enclose dedifferentiated and proliferating cardiomyocytes during regeneration. Induced genetic depletion of hapln1-expressing cells or genetic inactivation of hapln1b altered deposition of the key extracellular matrix component hyaluronic acid, disrupted cardiomyocyte proliferation, and inhibited heart regeneration. We also found that hapln1-expressing epicardial cells first emerge at the juvenile stage, when they associate with and are required for focused cardiomyocyte expansion events that direct maturation of the ventricular wall.Conclusions: Our findings identify a subset of epicardial cells that emerge in postembryonic zebrafish and sponsor regions of active cardiomyogenesis during cardiac growth and regeneration. We provide evidence that, as the heart achieves its mature structure, these cells facilitate hyaluronic acid deposition to support formation of the compact muscle layer of the ventricle. They are also required, along with the function of hapln1b paralog, in the production and organization of hyaluronic acid-containing matrix in cardiac injury sites, enabling normal cardiomyocyte proliferation and muscle regeneration. [ABSTRACT FROM AUTHOR]

Bioengineered hearts, which include single cardiomyocytes, engineered heart tissue, and chamber-like models, generate various biosignals, such as contractility, electrophysiological, and volume-pressure dynamic signals. Monitoring changes in these signals is crucial for understanding the mechanisms of disease progression and developing potential treatments. However, current methodologies face challenges in the continuous monitoring of bioengineered hearts over extended periods and typically require sacrificing the sample post-experiment, thereby limiting in-depth analysis. Thus, a biohybrid system consisting of living and nonliving components was developed. This system primarily features heart tissue alongside nonliving elements designed to support or comprehend its functionality. Biohybrid printing technology has simplified the creation of such systems and facilitated the development of various functional biohybrid systems capable of measuring or even regulating multiple functions, such as pacemakers, which demonstrates its versatility and potential applications. The future of biohybrid printing appears promising, with the ongoing exploration of its capabilities and potential directions for advancement., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Cardiac hypertrophy and its associated remodeling are among the leading causes of heart failure. Lysine crotonylation is a recently discovered posttranslational modification whose role in cardiac hypertrophy remains largely unknown. NAE1 (NEDD8-activating enzyme E1 regulatory subunit) is mainly involved in the neddylation modification of protein targets. However, the function of crotonylated NAE1 has not been defined. This study aims to elucidate the effects and mechanisms of NAE1 crotonylation on cardiac hypertrophy., Methods: Crotonylation levels were detected in both human and mouse subjects with cardiac hypertrophy through immunoprecipitation and Western blot assays. TMT-labeled quantitative lysine crotonylome analysis was performed to identify the crotonylated proteins in a mouse cardiac hypertrophic model induced by transverse aortic constriction. We generated NAE1 knock-in mice carrying a crotonylation-defective lysine to arginine K238R (lysine to arginine mutation at site 238) mutation (NAE1 K238R) and NAE1 knock-in mice expressing a crotonylation-mimicking lysine to glutamine K238Q (lysine to glutamine mutation at site 238) mutation (NAE1 K238Q) to assess the functional role of crotonylation of NAE1 at K238 in pathological cardiac hypertrophy. Furthermore, we combined coimmunoprecipitation, mass spectrometry, and dot blot analysis that was followed by multiple molecular biological methodologies to identify the target GSN (gelsolin) and corresponding molecular events contributing to the function of NAE1 K238 crotonylation., Results: The crotonylation level of NAE1 was increased in mice and patients with cardiac hypertrophy. Quantitative crotonylomics analysis revealed that K238 was the main crotonylation site of NAE1. Loss of K238 crotonylation in NAE1 K238R knock-in mice attenuated cardiac hypertrophy and restored the heart function, while hypercrotonylation mimic in NAE1 K238Q knock-in mice significantly enhanced transverse aortic constriction-induced pathological hypertrophic response, leading to impaired cardiac structure and function. The recombinant adenoviral vector carrying NAE1 K238R mutant attenuated, while the K238Q mutant aggravated Ang II (angiotensin II)-induced hypertrophy. Mechanistically, we identified GSN as a direct target of NAE1. K238 crotonylation of NAE1 promoted GSN neddylation and, thus, enhanced its protein stability and expression. NAE1 crotonylation-dependent increase of GSN promoted actin-severing activity, which resulted in adverse cytoskeletal remodeling and progression of pathological hypertrophy., Conclusions: Our findings provide new insights into the previously unrecognized role of crotonylation on nonhistone proteins during cardiac hypertrophy. We found that K238 crotonylation of NAE1 plays an essential role in mediating cardiac hypertrophy through GSN neddylation, which provides potential novel therapeutic targets for pathological hypertrophy and cardiac remodeling.

Competing Interests: Dr Diwan provides consulting services for Clario (previously ERT/Biomedical systems) and serves on the scientific advisory board for Dewpoint Therapeutics.

Background: β-adrenergic receptor (β-AR) overactivation is a major pathological cue associated with cardiac injury and diseases. AMPK (AMP-activated protein kinase), a conserved energy sensor, regulates energy metabolism and is cardioprotective. However, whether AMPK exerts cardioprotective effects via regulating the signaling pathway downstream of β-AR remains unclear., Methods: Using immunoprecipitation, mass spectrometry, site-specific mutation, in vitro kinase assay, and in vivo animal studies, we determined whether AMPK phosphorylates β-arrestin-1 at serine (Ser) 330. Wild-type mice and mice with site-specific mutagenesis (S330A knock-in [KI]/S330D KI) were subcutaneously injected with the β-AR agonist isoproterenol (5 mg/kg) to evaluate the causality between β-adrenergic insult and β-arrestin-1 Ser330 phosphorylation. Cardiac transcriptomics was used to identify changes in gene expression from β-arrestin-1-S330A/S330D mutation and β-adrenergic insult., Results: Metformin could decrease cAMP/PKA (protein kinase A) signaling induced by isoproterenol. AMPK bound to β-arrestin-1 and phosphorylated Ser330 with the highest phosphorylated mass spectrometry score. AMPK activation promoted β-arrestin-1 Ser330 phosphorylation in vitro and in vivo. Neonatal mouse cardiomyocytes overexpressing β-arrestin-1-S330D (active form) inhibited the β-AR/cAMP/PKA axis by increasing PDE (phosphodiesterase) 4 expression and activity. Cardiac transcriptomics revealed that the differentially expressed genes between isoproterenol-treated S330A KI and S330D KI mice were mainly involved in immune processes and inflammatory response. β-arrestin-1 Ser330 phosphorylation inhibited isoproterenol-induced reactive oxygen species production and NLRP3 (NOD-like receptor protein 3) inflammasome activation in neonatal mouse cardiomyocytes. In S330D KI mice, the β-AR-activated cAMP/PKA pathways were attenuated, leading to repressed inflammasome activation, reduced expression of proinflammatory cytokines, and mitigated macrophage infiltration. Compared with S330A KI mice, S330D KI mice showed diminished cardiac fibrosis and improved cardiac function upon isoproterenol exposure. However, the cardiac protection exerted by AMPK was abolished in S330A KI mice., Conclusions: AMPK phosphorylation of β-arrestin-1 Ser330 potentiated PDE4 expression and activity, thereby inhibiting β-AR/cAMP/PKA activation. Subsequently, β-arrestin-1 Ser330 phosphorylation blocks β-AR-induced cardiac inflammasome activation and remodeling., Competing Interests: None.

Background: Transverse (t)-tubules drive the rapid and synchronous Ca 2+ rise in cardiac myocytes. The virtual complete atrial t-tubule loss in heart failure (HF) decreases Ca 2+ release. It is unknown if or how atrial t-tubules can be restored and how this affects systolic Ca 2+ ., Methods: HF was induced in sheep by rapid ventricular pacing and recovered following termination of rapid pacing. Serial block-face scanning electron microscopy and confocal imaging were used to study t-tubule ultrastructure. Function was assessed using patchclamp, Ca 2+ , and confocal imaging. Candidate proteins involved in atrial t-tubule recovery were identified by western blot and expressed in rat neonatal ventricular myocytes to determine if they altered t-tubule structure., Results: Atrial t-tubules were lost in HF but reappeared following recovery from HF. Recovered t-tubules were disordered, adopting distinct morphologies with increased t-tubule length and branching. T-tubule disorder was associated with mitochondrial disorder. Recovered t-tubules were functional, triggering Ca 2+ release in the cell interior. Systolic Ca 2+ , I Ca-L , sarcoplasmic reticulum Ca 2+ content, and SERCA function were restored following recovery from HF. Confocal microscopy showed fragmentation of ryanodine receptor staining and movement away from the z-line in HF, which was reversed following recovery from HF. Acute detubulation, to remove recovered t-tubules, confirmed their key role in restoration of the systolic Ca 2+ transient, the rate of Ca 2+ removal, and the peak L-type Ca 2+ current. The abundance of telethonin and myotubularin decreased during HF and increased during recovery. Transfection with these proteins altered the density and structure of tubules in neonatal myocytes. Myotubularin had a greater effect, increasing tubule length and branching, replicating that seen in the recovery atria., Conclusions: We show that recovery from HF restores atrial t-tubules, and this promotes recovery of I Ca-L , sarcoplasmic reticulum Ca 2+ content, and systolic Ca 2+ . We demonstrate an important role for myotubularin in t-tubule restoration. Our findings reveal a new and viable therapeutic strategy.

Background: Drug-induced QT prolongation (diLQT) is a feared side effect that could expose susceptible individuals to fatal arrhythmias. The occurrence of diLQT is primarily attributed to unintended drug interactions with cardiac ion channels, notably the hERG (human ether-a-go-go-related gene) channels that generate the delayed-rectifier potassium current (I Kr ) and thereby regulate the late repolarization phase. There is an important interindividual susceptibility to develop diLQT, which is of unknown origin but can be reproduced in patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs). We aimed to investigate the dynamics of hERG channels in response to sotalol and to identify regulators of the susceptibility to developing diLQT., Methods: We measured electrophysiological activity and cellular distribution of hERG channels after hERG blocker treatment in iPS-CMs derived from patients with highest sensitivity (HS) or lowest sensitivity (LS) to sotalol administration in vivo (ie, on the basis of the measure of the maximal change in QT interval 3 hours after administration). Specific small interfering RNAs and CAVIN1-T2A-GFP adenovirus were used to manipulate CAVIN1 expression., Results: Whereas HS and LS iPS-CMs showed similar electrophysiological characteristics at baseline, the late repolarization phase was prolonged and I Kr significantly decreased after exposure of HS iPS-CMs to low sotalol concentrations. I Kr reduction was caused by a rapid translocation of hERG channel from the membrane to the cytoskeleton-associated fractions upon sotalol application. CAVIN1 , essential for caveolae biogenesis, was 2× more highly expressed in HS iPS-CMs, and its knockdown by small interfering RNA reduced their sensitivity to sotalol. CAVIN1 overexpression in LS iPS-CMs using adenovirus showed reciprocal effects. We found that treatment with sotalol promoted translocation of the hERG channel from the plasma membrane to the cytoskeleton fractions in a process dependent on CAVIN1 (caveolae associated protein 1) expression. CAVIN1 silencing reduced the number of caveolae at the membrane and abrogated the translocation of hERG channel in sotalol-treated HS iPS-CMs. CAVIN1 also controlled cardiomyocyte responses to other hERG blockers, such as E4031, vandetanib, and clarithromycin., Conclusions: Our study identifies unbridled turnover of the potassium channel hERG as a mechanism supporting the interindividual susceptibility underlying diLQT development and demonstrates how this phenomenon is finely tuned by CAVIN1., Competing Interests: Dr Hulot reports research grants from BioSerenity, Sanofi, Servier, and Novo Nordisk, and speaker, advisory board, or consultancy fees from Amgen, AstraZeneca, Bayer, BioSerenity, Bristol Myers Squibb, Novartis, Novo Nordisk, and Vifor Pharma, all unrelated to the present work. The other authors declare no competing financial interests.

Background: How the sarcomeric complex is continuously turned over in long-living cardiomyocytes is unclear. According to the prevailing model of sarcomere maintenance, sarcomeres are maintained by cytoplasmic soluble protein pools with free recycling between pools and sarcomeres., Methods: We imaged and quantified the turnover of expressed and endogenous sarcomeric proteins, including the giant protein titin, in cardiomyocytes in culture and in vivo, at the single cell and at the single sarcomere level using pulse-chase labeling of Halo-tagged proteins with covalent ligands., Results: We disprove the prevailing protein pool model and instead show an ordered mechanism in which only newly translated proteins enter the sarcomeric complex while older ones are removed and degraded. We also show that degradation is independent of protein age and that proteolytic extraction is a rate-limiting step in the turnover. We show that replacement of sarcomeric proteins occurs at a similar rate within cells and across the heart and is slower in adult cells., Conclusions: Our findings establish a unidirectional replacement model for cardiac sarcomeres subunit replacement and identify their turnover principles., Competing Interests: None.

In Canada and worldwide, the elderly population (ie, individuals > 65 years of age) is increasing disproportionately relative to the total population. This is expected to have a substantial impact on the health care system, as increased aged is associated with a greater incidence of chronic noncommunicable diseases. Within the elderly population, cardiovascular disease is a leading cause of death, therefore developing therapies that can prevent or slow disease progression in this group is highly desirable. Historically, aging research has focused on the development of anti-aging therapies that are implemented early in life and slow the age-dependent decline in cell and organ function. However, accumulating evidence supports that late-in-life therapies can also benefit the aged cardiovascular system by limiting age-dependent functional decline. Moreover, recent studies have demonstrated that rejuvenation (ie, reverting cellular function to that of a younger phenotype) of the already aged cardiovascular system is possible, opening new avenues to develop therapies for older individuals. In this review, we first provide an overview of the functional changes that occur in the cardiomyocyte with aging and how this contributes to the age-dependent decline in heart function. We then discuss the various anti-aging and rejuvenation strategies that have been pursued to improve the function of the aged cardiomyocyte, with a focus on therapies implemented late in life. These strategies include 1) established systemic approaches (caloric restriction, exercise), 2) pharmacologic approaches (mTOR, AMPK, SIRT1, and autophagy-targeting molecules), and 3) emerging rejuvenation approaches (partial reprogramming, parabiosis/modulation of circulating factors, targeting endogenous stem cell populations, and senotherapeutics). Collectively, these studies demonstrate the exciting potential and limitations of current rejuvenation strategies and highlight future areas of investigation that will contribute to the development of rejuvenation therapies for the aged heart., (Copyright © 2024 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.)

Background: Transplantation of human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) has emerged as a promising therapy to treat end-stage heart failure. However, the immunogenicity of hiPS-CMs in transplanted patients has not been fully elucidated. Thus, in vivo models are required to estimate immune responses against hiPS-CMs in transplant recipients., Methods: We transferred human peripheral blood mononuclear cells (hPBMCs) into NOD/Shi-scid IL-2rg null (NOG) MHC class I/II double knockout (NOG-ΔMHC) mice, which were reported to accept hPBMCs without xenogeneic-graft-versus-host disease (xeno-GVHD). Then, hiPS-CM sheets generated from the hiPS cell line 201B7 harboring a luciferase transgene were transplanted into the subcutaneous space of NOG-ΔMHC mice. Graft survival was monitored by bioluminescent images using a Xenogen In Vivo Imaging System., Results: The human immune cells were engrafted for more than 3 months in NOG-ΔMHC mice without lethal xeno-GVHD. The hiPS-CMs expressed a moderate level of human leukocyte antigen (HLA)-class I, but not HLA-class II, molecules even after interferon-gamma (IFN-γ) stimulation. Consistently, the allogenic IFN-γ-treated hiPS-CMs induced weak CD8 + but not CD4 + T cell responses in vitro. hiPS-CM sheets disappeared approximately 17 to 24 days after transplantation in hPBMC-transferred NOG-ΔMHC mice, and CD8 + T cell depletion significantly prolonged graft survival, similar to what was observed following tacrolimus treatment., Conclusions: hiPS-CMs are less immunogenic in vitro but induce sufficient CD8 + T cell-mediated immune responses for graft rejection in vivo., (Copyright © 2024 International Society for the Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Background: The mechanism of cardiac reverse remodeling (CRR) mediated by the left ventricular assist device remains unclear. This study aims to identify the specific cell type responsible for CRR and develop the therapeutic target that promotes CRR., Methods: The nuclei were extracted from the left ventricular tissue of 4 normal controls, 4 CRR patients, and 4 no cardiac reverse remodeling patients and then subjected to single-nucleus RNA sequencing for identifying key cell types responsible for CRR. Gene overexpression in transverse aortic constriction and dilated cardiomyopathy heart failure mouse model (C57BL/6J background) and pathological staining were performed to validate the results of single-nucleus RNA sequencing., Results: Ten cell types were identified among 126 156 nuclei. Cardiomyocytes in CRR patients expressed higher levels of ATP5F1A than the other 2 groups. The macrophages in CRR patients expressed more anti-inflammatory genes and functioned in angiogenesis. Endothelial cells that elevated in no cardiac reverse remodeling patients were involved in the inflammatory response. Echocardiography showed that overexpressing ATP5F1A through cardiomyocyte-specific adeno-associated virus 9 demonstrated an ability to improve heart function and morphology. Pathological staining showed that overexpressing ATP5F1A could reduce fibrosis and cardiomyocyte size in the heart failure mouse model., Conclusions: The present results of single-nucleus RNA sequencing and heart failure mouse model indicated that ATP5F1A could mediate CRR and supported the development of therapeutics for overexpressing ATP5F1A in promoting CRR., Competing Interests: None.

Background We previously, reported that granulocyte-colony stimulating factor (G-CSF) reduces cardiomyocyte apoptosis in diabetic cardiomyopathy. However, the underlying mechanisms are not yet fully understood. Therefore, we investigated whether the mechanisms underlying of the anti-apoptotic effects of G-CSF were associated with autophagy using a rat model of diabetic cardiomyopathy. Methods Diabetic cardiomyopathy was induced in rats through a high-fat diet combined with low-dose streptozotocin and the rats were then treated with G-CSF for 5 days. Rat H9c2 cardiac cells were cultured under high glucose conditions as an in vitro model of diabetic cardiomyopathy. The extent of apoptosis and protein levels related to autophagy (Beclin-1, microtubule-binding protein light chain 3 [LC3]-II/LC3-I ratio, and P62) were determined for both models. Autophagy determination was performed using an Autophagy Detection kit. Results G-CSF significantly reduced cardiomyocyte apoptosis in the diabetic myocardium in vivo and led to an increase in Beclin-1 level and the LC3-II/LC3-I ratio, and decreased P62 level. Similarly, G-CSF suppressed apoptosis, increased Beclin-1 level and LC3-II/LC3-I ratio, and decreased P62 level in high glucose-induced H9c2 cardiac cells in vitro. These effects of G-CSF were abrogated by 3-methyladenine, an autophagy inhibitor. In addition, G-CSF significantly increased autophagic flux in vitro. Conclusion Our results suggest that the anti-apoptotic effect of G-CSF might be significantly associated with the up-regulation of autophagy in diabetic cardiomyopathy.

E and F, Cluster analysis for single-nucleus RNA sequencing data from (E) all cells and (F) cardiomyocytes; results are presented according to (Ei and Fi) experimental group/time point, (Eii) cell type (defined by marker expression), (Eiii) abundance of each of 4 cardiomyocyte-specific genes, and (Fii) cardiomyocyte cluster. The cardiomyocyte 1 cluster included cardiomyocytes from all other animal groups and time points as well as a small number of AR SB P1 sb MI SB P28 sb SB -P35 sb cardiomyocytes but few (<1%) fetal or control-P56 cardiomyocytes (Figure [G]). [Extracted from the article]

Background: Human pluripotent stem cell (hPSC)-derived cardiomyocytes (hPSC-CMs) have tremendous promise for application in cardiac regeneration, but their translational potential is limited by an immature phenotype. We hypothesized that large-scale manufacturing of mature hPSC-CMs could be achieved through culture on polydimethylsiloxane (PDMS)-lined roller bottles and that the transplantation of these cells would mediate better structural and functional outcomes than with conventional immature hPSC-CM populations.Methods: We comprehensively phenotyped hPSC-CMs after in vitro maturation for 20 and 40 days on either PDMS or standard tissue culture plastic substrates. All hPSC-CMs were generated from a transgenic hPSC line that stably expressed a voltage-sensitive fluorescent reporter to facilitate in vitro and in vivo electrophysiological studies, and cardiomyocyte populations were also analyzed in vitro by immunocytochemistry, ultrastructure and fluorescent calcium imaging, and bulk and single-cell transcriptomics. We next compared outcomes after the transplantation of these populations into a guinea pig model of myocardial infarction using end points including histology, optical mapping of graft- and host-derived action potentials, echocardiography, and telemetric electrocardiographic monitoring.Results: We demonstrated the economic generation of >1×108 mature hPSC-CMs per PDMS-lined roller bottle. Compared with their counterparts generated on tissue culture plastic substrates, PDMS-matured hPSC-CMs exhibited increased cardiac gene expression and more mature structural and functional properties in vitro. More important, intracardiac grafts formed with PDMS-matured myocytes showed greatly enhanced structure and alignment, better host-graft electromechanical integration, less proarrhythmic behavior, and greater beneficial effects on contractile function.Conclusions: We describe practical methods for the scaled generation of mature hPSC-CMs and provide the first evidence that the transplantation of more mature cardiomyocytes yields better outcomes in vivo. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of long non-coding RNA insulin-like growth factor 2 antisense transcript (lncRNA IGF2-AS) on the cardiomyocyte-like differentiation of bone marrow mesenchymal stem cells (BMSCs) by regulating insulin-like growth factor 2 (IGF2). Methods BMSCs were isolated and cultured from SD rats. The morphology of primary and third-generation cells was observed under inverted microscope. Flow cytometry was used to detect the expressions of CD29, CD90 and CD45 on the surface of BMSCs. BMSCs with good growth in the third generation were divided into the control group, the empty vector group (transfected with pLVX-IRES-Zs Green1 vector), the lncRNA IGF2- AS group (transfected with pLVX-IRES-Zs Green1-IGF2-AS), the 5-azacytosine group (8 μmol/L 5-AZA treatment), the si-NC group, the si-IGF2-AS group, the si-IGF2 group, lncRNA IGF2-AS+si-NC group and the lncRNA IGF2-AS+si- IGF2 group. Quantificational real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of IGF2- AS. MTT method was used to detect cell viability. Western blot assay was used to detect the IGF2, gap junction protein 43 (Cx43), cardiac troponin T (cTnT) and cardiac troponin I (cTnI) protein expression. RNA pull-down and RNA immunoprecipitation (RIP) were used to detect the IGF2-AS and IGF2 protein binding. Results The primary cells were in suspension at the beginning of the culture flask, most of them were round, and they began to grow adherently after 48 h of culture. The third-generation cells were long spindle-shaped, irregularly arranged, closely connected between adjacent cells, and fibroblast-like. The third-generation BMSCs showed high expression of CD29 (98.21%), CD90 (92.54%), and low expression of CD45 (3.67%). The overexpression of IGF2-AS or 5-AZA could promote protein expressions of Cx43, cTnT and cTnI in BMSCs, and reduce cell viability, and the corresponding indicators were significantly lower in the 5-AZA group than those in the lncRNA IGF2-AS group (P＜0.05). Silencing IGF2-AS or inhibiting the expression of IGF2 could reduce the protein expressions of Cx43, cTnT, and cTnI in BMSCs, and reduce cell viability (P＜0.05). lncRNA IGF2-AS could target up-regulate the expression of IGF2 protein (P＜0.05). Silencing IGF2 reversed the promotion effect of over-expression of IGF2-AS on cardiomyocyte-like differentiation of BMSCs. Conclusion Overexpression of IGF2-AS promotes cardiomyocyte-like differentiation of BMSCs by up-regulating IGF2. [ABSTRACT FROM AUTHOR]

Objective To investigate the effects of secretory clusterin (sCLU) on oxidative-induced cardiomyocyte injury and the underlying mechanism. Methods Rat cardiomyocytes H9C2 were used in this experiment. Experiment 1 was divided into the control group (only medium change) and the H2O2 group (treated with 100 µmol/L H2O2). Experiment 2 was divided into the control group, the pcDNA3.1 group (transfected with pcDNA3.1 empty plasmids), the pcDNA3.1-sCLU group (transfected with pcDNA3.1-sCLU overexpressed plasmids), the H2O2 group and the H2O2+pcDNA3.1 group (transfected with pcDNA3.1 empty plasmids for 48 h, and then treated with 100 µmol/L H2O2). The H2O2+pcDNA3.1-sCLU was transfected with pcDNA3.1-sCLU over-expressed plasmids for 48 h, and then 100 µmol/L H2O2. Experiment 3 was divided into the DMSO group (adding 1% volume of DMSO), the Mdivi-1 group (treated with 10 µmol/L Mdivi-1), the H2O2+DMSO group (treated with 1% volume of DMSO for 30 min, and then added 100 µmol/L H2O2), the H2O2+Mdivi-1 group (treated with 100 µmol/L Mdivi-1 for 30 min, and then treated with 100 µmol/L H2O2), the H2O2+pcDNA3.1-sCLU group and the H2O2+pcDNA3.1-sCLU+Mdivi-1 group (transfected with pcDNA3.1-sCLU overexpressed plasmids for 48 h, and then treated with 10 µmol/L Mdivi-1 for 30 min, and then added 100 µmol/L H2O2). MTT assay was used to detect cell viability. TUNEL assay was used to detect cell apoptosis. The corresponding Kit was used to detect the activity of reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA). The expression of sCLU in cells was detected by qRT-PCR and ELISA. The protein expression levels of CLU, mitochondrial autophagy related proteins PINK1, Parkin and LC3 were detected by Western blot assay. Results In experiment 1, compared with the control group, the relative expression levels of sCLU mRNA, sCLU protein levels in cell culture supernatant and total CLU protein levels in cells were decreased in the H2O2 group (P＜0.05). In experiment 2, compared with the control group, cell viability, SOD levels and LC3-II/LC3-I ratio were all decreased in the H2O2 group (P＜0.05). Apoptosis rate, ROS and MDA levels, PINK1 and Parkin protein expression levels were all increased in the H2O2 group (P＜0.05). Compared with H2O2 group, cell viability rate, SOD levels and LC3-Ⅱ/LC3-Ⅰ ratio were increased in H2O2+pcDNA3.1-sCLU group (P＜0.05). Apoptosis rate, ROS and MDA levels, PINK1 and Parkin protein expression levels were all decreased in H2O2+pcDNA3.1-sCLU group (P＜0.05). In experiment 3, compared with DMSO group, mdivi-1 group showed no significant difference in cell viability and apoptosis rate. Cell viability was decreased and apoptosis rate was increased in H2O2+DMSO group (P＜0.05). Compared with the H2O2+DMSO group, cell viability was increased and cell apoptosis rate was decreased in the H2O2+ Mdivi-1 group and H2O2+pcDNA3.1-sCLU group (P＜0.05). There were no significant differences in cell viability and the cell apoptosis rate between the DMSO group and the Mdivi-1 group. Compared with the DMSO group, the cell viability decreased and the cell apoptosis rate increased in the H2O2+DMSO group (P＜0.05). Compared with the H2O2+DMSO group, cell viability increased and cell apoptosis rate decreased in the H2O2+Mdivi-1 group, the H2O2+PCDNA3.1-sCLU group and the H2O2+PCDNA3.1-SCLU+Mdivi-1 group (P＜0.05). There was no significant difference in cell viability between the H2O2+Mdivi-1 group, H2O2+pcDNA3.1-sCLU group and the H2O2+pcDNA3.1-sCLU+Mdivi-1 group (P＞ 0.05). Conclusion sCLU can protect myocardial cells under oxidative stress, and the mechanism may be related to the inhibition of mitophagy. [ABSTRACT FROM AUTHOR]

Bradyarrhythmia, a life-threatening cardiovascular disease, is an increasing burden for the healthcare system. Currently, surgery, implanted device, and drug are introduced to treat the bradyarrhythmia in clinical practice. However, these conventional therapeutic strategies suffer from the invasive surgery, power supply, or drug side effect, respectively, hence developing the alternative therapeutic strategy is necessarily imperative. Here, a convenient and effective strategy to treat the bradyarrhythmia is proposed using near-infrared-triggered Au nanorod (NR) based plasmonic photothermal effect (PPE). Moreover, electrophysiology of cardiomyocytes is dynamically monitored by the integrated biosensing-regulating system during and after the treatment. Cardiomyocyte-based bradyarrhythmia recover rhythmic for a long time by regulating plasmonic photothermal effect. Furthermore, the regulatory mechanism is qualitatively investigated to verify the significant thermal stimulation in the recovery process. This study establishes a reliable platform for long-term recording and evaluation of mild photothermal therapy for bradyarrhythmia in vitro, offering an efficient and non-invasive strategy for the potential clinical applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

To investigate the role of silent mating type information regulator 2 homolog 3 (SIRT3) - forkhead box O1(FOXO1) pathway-mediated autophagy in myocardial ischemia-reperfusion (IR) injury. Methods SIRT3 overexpression stable strain was constructed by lentivirus infection of H9C2 cells. The cells were divided into four groups: the normal control (NC) group, the ischemia-reperfusion (IR) group, the SIRT3 overexpression +IR (S+IR) group and the SIRT3 overexpression +IR+ SIRT3 inhibitor (S+IR+3-TYP) group. The expressions of SIRT3, Ac-FOXO1, autophagy labeled protein LC3B, Beclin1 and apoptotic proteins Bcl-2 and Bax were detected by Western blot assay. The apoptosis rate was detected by flow cytometry. The level of lactate dehydrogenase (LDH) in the supernatant of cell culture was determined by automatic biochemical analyzer. Results Compared with the NC group, the expression of SIRT3 was decreased, AcFOXO1 was increased, the expressions of autophagy related proteins Beclin1 and LC3B were increased, the expression of antiapoptotic protein Bcl-2 was decreased, the expression of pro-apoptotic protein Bax was increased, LDH release and cell apoptosis rate were increased in the IR group (P＜0.05). Compared with the IR group, the expression of SIRT3 increased, Beclin1, LC3B and Bcl-2 were increased, the expressions of Ac-FOXO1, pro-apoptotic protein Bax were decreased, LDH release and cell apoptosis rate were decreased in the S+IR group (P＜0.05). After adding SIRT3 inhibitor 3-TYP, there was no significant change in SIRT3 expression compared with that of the S+IR group, but the expression of Ac-FOXO1 was increased, the expressions of Beclin1 and LC3B were decreased, the expression of anti-apoptotic protein Bcl-2 was decreased, the expression of pro-apoptotic protein Bax was increased, LDH release and cell apoptosis rate were increased (P＜0.05). Conclusion The activation of SIRT3-FOXO1 pathway can increase autophagy level of H9C2 cells, and has a protective effect on myocardial ischemia reperfusion injury. [ABSTRACT FROM AUTHOR]

Supplemental Digital Content is available in the text. Background: Inflammation contributes to the pathogenesis of heart failure, but there is limited understanding of inflammation's potential benefits. Inflammatory cells secrete MYDGF (myeloid-derived growth factor) to promote tissue repair after acute myocardial infarction. We hypothesized that MYDGF has a role in cardiac adaptation to persistent pressure overload. Methods: We defined the cellular sources and function of MYDGF in wild-type (WT), Mydgf -deficient (Mydgf −/−), and Mydgf bone marrow–chimeric or bone marrow–conditional transgenic mice with pressure overload–induced heart failure after transverse aortic constriction surgery. We measured MYDGF plasma concentrations by targeted liquid chromatography–mass spectrometry. We identified MYDGF signaling targets by phosphoproteomics and substrate-based kinase activity inference. We recorded Ca2+ transients and sarcomere contractions in isolated cardiomyocytes. Additionally, we explored the therapeutic potential of recombinant MYDGF. Results: MYDGF protein abundance increased in the left ventricular myocardium and in blood plasma of pressure-overloaded mice. Patients with severe aortic stenosis also had elevated MYDGF plasma concentrations, which declined after transcatheter aortic valve implantation. Monocytes and macrophages emerged as the main MYDGF sources in the pressure-overloaded murine heart. While Mydgf −/− mice had no apparent phenotype at baseline, they developed more severe left ventricular hypertrophy and contractile dysfunction during pressure overload than WT mice. Conversely, conditional transgenic overexpression of MYDGF in bone marrow–derived inflammatory cells attenuated pressure overload–induced hypertrophy and dysfunction. Mechanistically, MYDGF inhibited G protein–coupled receptor agonist–induced hypertrophy and augmented SERCA2a (sarco/endoplasmic reticulum Ca2+-ATPase 2a) expression in cultured neonatal rat ventricular cardiomyocytes by enhancing PIM1 (Pim-1 proto-oncogene, serine/threonine kinase) expression and activity. Along this line, cardiomyocytes from pressure-overloaded Mydgf −/− mice displayed reduced PIM1 and SERCA2a expression, greater hypertrophy, and impaired Ca2+ cycling and sarcomere function compared with cardiomyocytes from pressure-overloaded WT mice. Transplanting Mydgf −/− mice with WT bone marrow cells augmented cardiac PIM1 and SERCA2a levels and ameliorated pressure overload–induced hypertrophy and dysfunction. Pressure-overloaded Mydgf −/− mice were similarly rescued by adenoviral Serca2a gene transfer. Treating pressure-overloaded WT mice subcutaneously with recombinant MYDGF enhanced SERCA2a expression, attenuated left ventricular hypertrophy and dysfunction, and improved survival. Conclusions: These findings establish a MYDGF-based adaptive crosstalk between inflammatory cells and cardiomyocytes that protects against pressure overload–induced heart failure. [ABSTRACT FROM AUTHOR]

Cardiac hypertrophy characterized by abnormal cardiomyocyte viscosity is a typical sign of heart failure (HF) with vital importance for early diagnosis. However, current biochemical and imaging diagnostic methods are unable to detect this subclinical manifestation. In this work, we developed a series of NIR-I fluorescence probes for detecting myocardial viscosity based on the pyridazinone scaffold. The probes showed weak fluorescence due to free intramolecular rotation under low-viscosity conditions, while they displayed strong fluorescence with limited intramolecular rotation in response to a high-viscosity environment. Among them, CarVis2 exhibited higher stability and photobleaching resistance than commercial dyes. Its specific response to viscosity was not influenced by the pH and biological species. Furthermore, CarVis2 showed rapid and accurate responses to the viscosity of isoproterenol (ISO)-treated H9C2 cardiomyocytes with good biocompatibility. More importantly, CarVis2 demonstrated excellent sensitivity in monitoring myocardial viscosity variation in HF mice in vivo, potentially enabling earlier noninvasive identification of myocardial abnormalities compared to traditional clinical imaging and biomarkers. These findings revealed that CarVis2 can serve as a powerful tool to monitor myocardial viscosity, providing the potential to advance insights into a pathophysiological mechanism and offering a new reference strategy for early visual diagnosis of HF.

Background: Calcium (Ca 2+ ) uptake by mitochondria occurs via the mitochondrial Ca 2+ uniporter. Mitochondrial Ca 2+ uniporter exists as a complex, regulated by 3 MICU (mitochondrial Ca 2+ uptake) proteins localized in the intermembrane space: MICU1, MICU2, and MICU3. Although MICU3 is present in the heart, its role is largely unknown., Methods: We used CRISPR-Cas9 to generate a mouse with global deletion of MICU3 and an adeno-associated virus (AAV9) to overexpress MICU3 in wild-type mice. We examined the role of MICU3 in regulating mitochondrial calcium ([Ca 2+ ] m ) in ex vivo hearts using an optical method following adrenergic stimulation in perfused hearts loaded with a Ca 2+ -sensitive fluorophore. Additionally, we studied how deletion and overexpression of MICU3, respectively, impact cardiac function in vivo by echocardiography and the molecular composition of the mitochondrial Ca 2+ uniporter complex via Western blot, immunoprecipitation, and Blue native-PAGE analysis. Finally, we measured MICU3 expression in failing human hearts., Results: MICU3 knock out hearts and cardiomyocytes exhibited a significantly smaller increase in [Ca 2+ ] m than wild-type hearts following acute isoproterenol infusion. In contrast, heart with overexpression of MICU3 exhibited an enhanced increase in [Ca 2+ ] m compared with control hearts. Echocardiography analysis showed no significant difference in cardiac function in knock out MICU3 mice relative to wild-type mice at baseline. However, mice with overexpression of MICU3 exhibited significantly reduced ejection fraction and fractional shortening compared with control mice. We observed a significant increase in the ratio of heart weight to tibia length in hearts with overexpression of MICU3 compared with controls, consistent with hypertrophy. We also found a significant decrease in MICU3 protein and expression in failing human hearts., Conclusions: Our results indicate that increased and decreased expression of MICU3 enhances and reduces, respectively, the uptake of [Ca 2+ ] m in the heart. We conclude that MICU3 plays an important role in regulating [Ca 2+ ] m physiologically, and overexpression of MICU3 is sufficient to induce cardiac hypertrophy, making MICU3 a possible therapeutic target., Competing Interests: Disclosures None.