Cardiomyocyte ßII spectrin plays a critical role in maintaining cardiac function by regulating mitochondrial respiratory function.

AIMS: ßII spectrin is a cytoskeletal protein known to be tightly linked to heart development and cardiovascular electrophysiology. However, the roles of ßII spectrin in cardiac contractile function and pathological post-myocardial infarction remodeling remain unclear. Here, we investigated whether and how ßII spectrin, the most common isoform of non-erythrocytic spectrin in cardiomyocytes, is involved in cardiac contractile function and ischemia/reperfusion (I/R) injury. METHODS AND RESULTS: We observed that the levels of serum ßII spectrin breakdown products (ßII SBDPs) were significantly increased in patients with acute myocardial infarction (AMI). Concordantly, ßII spectrin was degraded into ßII SBDPs by calpain in mouse hearts after I/R injury. Using tamoxifen-inducible cardiac-specific ßII spectrin knockout mice, we found that deletion of ßII spectrin in the adult heart resulted in spontaneous development of cardiac contractile dysfunction, cardiac hypertrophy and fibrosis at 5 weeks after tamoxifen treatment. Moreover, at 1 week after tamoxifen treatment, although spontaneous cardiac dysfunction in cardiac-specific ßII spectrin knockout mice had not developed, deletion of ßII spectrin in the heart exacerbated I/R-induced cardiomyocyte death and heart failure. Furthermore, restoration of ßII spectrin expression via adenoviral small activating RNA (saRNA) delivery into the heart reduced I/R injury. Immunoprecipitation coupled with mass spectrometry (IP-LC-MS/MS) analyses and functional studies revealed that ßII spectrin is indispensable for mitochondrial complex I activity and respiratory function. Mechanistically, ßII spectrin promotes translocation of NADH:ubiquinone oxidoreductase 75 kDa Fe-S protein 1 (NDUFS1) from the cytosol to mitochondria by crosslinking with actin filaments (F-actin) to maintain F-actin stability. CONCLUSION: ßII spectrin is an essential cytoskeletal element for preserving mitochondrial homeostasis and cardiac function. Defects in ßII spectrin exacerbate cardiac I/R injury.

Extracellular Vesicles in Cardiovascular Pathophysiology: Communications, Biomarkers, and Therapeutic Potential.

Extracellular vesicles (EVs) are diverse, membrane-bound vesicles released from cells into the extracellular environment. They originate from either endosomes or the cell membrane and typically include exosomes and microvesicles. These EVs serve as crucial mediators of intercellular communication, carrying a variety of contents such as nucleic acids, proteins, and lipids, which regulate the physiological and pathological processes of target cells. Moreover, the molecular cargo of EVs can reflect critical information about the originating cells, making them potential biomarkers for the diagnosis and prognosis of diseases. Over the past decade, the role of EVs as key communicators between cell types in cardiovascular physiology and pathology has gained increasing recognition. EVs from different cellular sources, or from the same source under different cellular conditions, can have distinct impacts on the management, diagnosis, and prognosis of cardiovascular diseases. Furthermore, it is essential to consider the influence of cardiovascular-derived EVs on the metabolism of peripheral organs. This review aims to summarize recent advancements in the field of cardiovascular research with respect to the roles and implications of EVs. Our goal is to provide new insights and directions for the early prevention and treatment of cardiovascular diseases, with an emphasis on the therapeutic potential and diagnostic value of EVs.

Rectifying METTL4-Mediated N6-Methyladenine Excess in Mitochondrial DNA Alleviates Heart Failure.

BACKGROUND: Myocardial mitochondrial dysfunction underpins the pathogenesis of heart failure (HF), yet therapeutic options to restore myocardial mitochondrial function are scarce. Epigenetic modifications of mitochondrial DNA (mtDNA), such as methylation, play a pivotal role in modulating mitochondrial homeostasis. However, their involvement in HF remains unclear. METHODS: Experimental HF models were established through continuous angiotensin II and phenylephrine (AngII/PE) infusion or prolonged myocardial ischemia/reperfusion injury. The landscape of N6-methyladenine (6mA) methylation within failing cardiomyocyte mtDNA was characterized using high-resolution mass spectrometry and methylated DNA immunoprecipitation sequencing. A tamoxifen-inducible cardiomyocyte-specific Mettl4 knockout mouse model and adeno-associated virus vectors designed for cardiomyocyte-targeted manipulation of METTL4 (methyltransferase-like protein 4) expression were used to ascertain the role of mtDNA 6mA and its methyltransferase METTL4 in HF. RESULTS: METTL4 was predominantly localized within adult cardiomyocyte mitochondria. 6mA modifications were significantly more abundant in mtDNA than in nuclear DNA. Postnatal cardiomyocyte maturation presented with a reduction in 6mA levels within mtDNA, coinciding with a decrease in METTL4 expression. However, an increase in both mtDNA 6mA level and METTL4 expression was observed in failing adult cardiomyocytes, suggesting a shift toward a neonatal-like state. METTL4 preferentially targeted mtDNA promoter regions, which resulted in interference with transcription initiation complex assembly, mtDNA transcriptional stalling, and ultimately mitochondrial dysfunction. Amplifying cardiomyocyte mtDNA 6mA through METTL4 overexpression led to spontaneous mitochondrial dysfunction and HF phenotypes. The transcription factor p53 was identified as a direct regulator of METTL4 transcription in response to HF-provoking stress, thereby revealing a stress-responsive mechanism that controls METTL4 expression and mtDNA 6mA. Cardiomyocyte-specific deletion of the Mettl4 gene eliminated mtDNA 6mA excess, preserved mitochondrial function, and mitigated the development of HF upon continuous infusion of AngII/PE. In addition, specific silencing of METTL4 in cardiomyocytes restored mitochondrial function and offered therapeutic relief in mice with preexisting HF, irrespective of whether the condition was induced by AngII/PE infusion or myocardial ischemia/reperfusion injury. CONCLUSIONS: Our findings identify a pivotal role of cardiomyocyte mtDNA 6mA and the corresponding methyltransferase, METTL4, in the pathogenesis of mitochondrial dysfunction and HF. Targeted suppression of METTL4 to rectify mtDNA 6mA excess emerges as a promising strategy for developing mitochondria-focused HF interventions.

Knockdown of BCL-3 Attenuates Inflammatory Response in Intracerebral Hemorrhage Through an rBMECs/MGs Microenvironment.

Intracerebral hemorrhage (ICH) is a severe disease with high mortality. Recently, the role of BCL-3 in ICH has started to gain attention, but its mechanism remains unclear. A collagenase injection method was used to establish an ICH model in rats, and the expression of BCL-3 were detected. Rat brain microvascular endothelial cells (rBMECs) were isolated and induced with Hemin to establish an in vitro ICH model. The expression of BCL-3 was assessed, followed by detection of cell apoptosis. In the cell model, the recruitment, polarization, and pro-inflammatory features of the microglia (MGs) were assessed after co-cultured with rBMECs. Finally, in the ICH animal model, after knockdown of BCL-3, comprehensive evaluations of inflammatory responses in brain tissue, polarization and recruitment of microglia, and apoptosis were conducted. Results revealed an upregulated expression of BCL-3 in brain tissue of the ICH animal model. In Hemin-treated rBMECs, an upward trend in BCL-3 expression was observed, accompanied by an increase of cell apoptosis. After co-culturing with the in vitro model, microglia exhibited enhanced M1 polarization and intensified inflammatory responses. However, when BCL-3 expression was inhibited in the in vitro model, a reversal occurred in the polarization tendency and inflammatory responses of microglia. Additionally, after knockdown of BCL-3 in the animal model, notable improvements occurred in M1 polarization, infiltration of macrophages, and inflammatory reactions in the brain tissue. Therefore, BCL-3 modulates the inflammatory response after ICH occurrence through the BMECs/MGs microenvironment. Additionally, BCL-3 might be a potential therapeutic target for ICH management.

Identifying Obstructive Hypertrophic Cardiomyopathy from Nonobstructive Hypertrophic Cardiomyopathy: Development and Validation of a Model Based on Electrocardiogram Features.

Background: The clinical presentation and prognosis of hypertrophic cardiomyopathy (HCM) are heterogeneous between nonobstructive HCM (HNCM) and obstructive HCM (HOCM). Electrocardiography (ECG) has been used as a screening tool for HCM. However, it is still unclear whether the features presented on ECG could be used for the initial classification of HOCM and HNCM. Objective: We aimed to develop a pragmatic model based on common 12-lead ECG features for the initial identification of HOCM/HNCM. Methods: Between April 1st and September 30th, 2020, 172 consecutive HCM patients from the International Cooperation Center for Hypertrophic Cardiomyopathy of Xijing Hospital were prospectively included in the training cohort. Between January 4th and February 30th, 2021, an additional 62 HCM patients were prospectively included in the temporal internal validation cohort. External validation was performed using retrospectively collected ECG data with definite classification (390 HOCM and 499 HNCM ECG samples) from January 1st, 2010 to March 31st, 2020. Multivariable backward logistic regression (LR) was used to develop the prediction model. The discrimination performance, calibration and clinical utility of the model were evaluated. Results: Of all 30 acquired ECG parameters, 10 variables were significantly different between HOCM and HNCM (all P < 0.05). The P wave interval and SV1 were selected to construct the model, which had a clearly useful C-statistic of 0.805 (0.697, 0.914) in the temporal validation cohort and 0.776 (0.746, 0.806) in the external validation cohort for differentiating HOCM from HNCM. The calibration plot, decision curve analysis, and clinical impact curve indicated that the model had good fitness and clinical utility. Conclusion: The pragmatic model constructed by the P wave interval and SV1 had a clearly useful ability to discriminate HOCM from HNCM. The model might potentially serve as an initial classification of HCM before referring patients to dedicated centers and specialists. Highlights: What are the novel findings of this work? Evident differences exist in the ECG presentations between HOCM and HNCM.To the best of our knowledge, this study is the first piece of evidence to quantify the difference in the ECG presentations between HOCM and HNCM.Based on routine 12-lead ECG data, a probabilistic model was generated that might assist in the initial classification of HCM patients.

Characterization of the circulating transcriptome expression profile and identification of novel miRNA biomarkers in hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM), one of the most common genetic cardiovascular diseases, but cannot be explained by single genetic factors. Circulating microRNAs (miRNAs) are stable and highly conserved. Inflammation and immune response participate in HCM pathophysiology, but whether the miRNA profile changes correspondingly in human peripheral blood mononuclear cells (PBMCs) with HCM is unclear. Herein, we aimed to investigate the circulating non-coding RNA (ncRNA) expression profile in PBMCs and identify potential miRNAs for HCM biomarkers. METHODS: A Custom CeRNA Human Gene Expression Microarray was used to identify differentially expressed (DE) mRNAs, miRNAs, and ncRNAs (including circRNA and lncRNA) in HCM PBMCs. Weighted correlation network analysis (WGCNA) was used to identify HCM-related miRNA and mRNA modules. The mRNAs and miRNAs from the key modules were used to construct a co-expression network. Three separate machine learning algorithms (random forest, support vector machine, and logistic regression) were applied to identify potential biomarkers based on miRNAs from the HCM co-expression network. Gene Expression Omnibus (GEO) database (GSE188324) and experimental samples were used for further verification. Gene set enrichment analysis (GSEA) and competing endogenous RNA (ceRNA) network was used to determine the potential functions of the selected miRNAs in HCM. RESULTS: We identified 1194 DE-mRNAs, 232 DE-miRNAs and 7696 DE-ncRNAs in HCM samples compared with normal controls from the microarray data sets. WGCNA identified key miRNA modules and mRNA modules evidently associated with HCM. We constructed a miRNA‒mRNA co-expression network based on these modules. A total of three hub miRNAs (miR-924, miR-98 and miR-1) were identified by random forest, and the areas under the receiver operator characteristic curves of miR-924, miR-98 and miR-1 were 0.829, 0.866, and 0.866, respectively. CONCLUSIONS: We elucidated the transcriptome expression profile in PBMCs and identified three hub miRNAs (miR-924, miR-98 and miR-1) as potential biomarkers for HCM detection.

Suppressing Mesenchymal Stromal Cell Ferroptosis Via Targeting a Metabolism-Epigenetics Axis Corrects their Poor Retention and Insufficient Healing Benefits in the Injured Liver Milieu.

Mesenchymal stromal cell (MSC) implantation is a promising option for liver repair, but their poor retention in the injured liver milieu critically blunts therapeutic effects. The aim is to clarify the mechanisms underlying massive MSC loss post-implantation and establish corresponding improvement strategies. MSC loss primarily occurs within the initial hours after implantation into the injured liver milieu or under reactive oxygen species (ROS) stress. Surprisingly, ferroptosis is identified as the culprit for rapid depletion. In ferroptosis- or ROS-provoking MSCs, branched-chain amino acid transaminase-1 (BCAT1) is dramatically decreased, and its downregulation renders MSC susceptible to ferroptosis via suppressing the transcription of glutathione peroxidase-4 (GPX4), a vital ferroptosis defensing enzyme. BCAT1 downregulation impedes GPX4 transcription via a rapid-responsive metabolism-epigenetics coordinating mechanism, involving α-ketoglutarate accumulation, histone 3 lysine 9 trimethylation loss, and early growth response protein-1 upregulation. Approaches to suppress ferroptosis (e.g., incorporating ferroptosis inhibitors in injection solvent and overexpressing BCAT1) significantly improve MSC retention and liver-protective effects post-implantation. This study provides the first evidence indicating that excessive MSC ferroptosis is the nonnegligible culprit for their rapid depletion and insufficient therapeutic efficacy after implantation into the injured liver milieu. Strategies suppressing MSC ferroptosis are conducive to optimizing MSC-based therapy.

Variation of nitrate and nitrite in condensable particulate matter from coal-fired power plants under the simulated rapid condensing conditions.

In this work, condensation temperature, H2O vapor, SO2, SO3 and NH3 were studied to explore the formation mechanism of nitrate ions (NO3-) and nitrite ions (NO2-) in condensable particulate matter (CPM) discharged by ultra-low emission coal-fired power plants. Some important results were obtained: (i) The concentration of NO3- and NO2- increased with the decrease of condensation temperature, and H2O vapor could also promote the formation of NO3- and NO2-. (ii) The effects of SO2 and SO3 varied at different saturated states of flue gas, which was caused by the redox reaction of SO2 and NOX or the formation of H2SO4. (iii) NH3 could promote the nucleation of NO3- and NO2-, and the promotion effect also existed in the existence of SO2 or SO3. It is worth mentioning that SO3 and SO2 might synergistically inhibit the formation of NO3- and NO2-, regardless of the presence of NH3. The research results would enrich peoples understanding of the chemical and physical characteristics of NO3- and NO2- in CPM and provide a basic reference for the control of CPM emitted from coal-fired power plants.

Upregulation of GLT25D1 in Hepatic Stellate Cells Promotes Liver Fibrosis via the TGF-ß1/SMAD3 Pathway In Vivo and In vitro.

Background and Aims: Collagen ß(1-O) galactosyltransferase 25 domain 1 (GLT25D1) is associated with collagen production and glycosylation, and its knockout in mice results in embryonic death. However, its role in liver fibrosis remains elusive, particularly in hepatic stellate cells (HSCs), the primary collagen-producing cells associated with liver fibrogenesis. Herein, we aimed to elucidate the role of GLT25D1 in HSCs. Methods: Bile duct ligation (BDL)-induced mouse liver fibrosis models, primary mouse HSCs (mHSCs), and transforming growth factor beta 1 (TGF-ß1)-stimulated LX-2 human hepatic stellate cells were used in in vivo and in vitro studies. Stable LX-2 cell lines with either GLT25D1 overexpression or knockdown were established using lentiviral transfection. RNA-seq was performed to investigate the genomic differences. HPLC-MS/MS were used to identify glycosylation sites. Scanning electronic microscopy (SEM) and second-harmonic generation/two-photon excited fluorescence (SHG/TPEF) were used to image collagen fibril morphology. Results: GLT25D1 expression was upregulated in nonparenchymal cells in human cirrhotic liver tissues. Meanwhile, its knockdown attenuated collagen deposition in BDL-induced mouse liver fibrosis and inhibited mHSC activation. GLT25D1 was overexpressed in activated versus quiescence LX-2 cells and regulated in vitro LX-2 cell activation, including proliferation, contraction, and migration. GLT25D1 also significantly increased liver fibrogenic gene and protein expression. GLT25D1 upregulation promoted HSC activation and enhanced collagen expression through the TGF-ß1/SMAD signaling pathway. Mass spectrometry showed that GLT25D1 regulated the glycosylation of collagen in HSCs, affecting the diameter of collagen fibers. Conclusions: Collectively, the upregulation of GLT25D1 in HSCs promoted the progression of liver fibrosis by affecting HSCs activation and collagen stability.

Physicochemical characteristics of polycyclic aromatic hydrocarbons in condensable particulate matter from coal-fired power plants: A laboratory simulation.

The objective of this study was to examine the physicochemical characteristics of polycyclic aromatic hydrocarbons (PAHs) in condensable particulate matter (CPM) during fast condensation (within several seconds). The concentration of PAHs increased as the condensation temperature decreased, indicating that the conversion of gaseous PAHs to CPM would be enhanced at low temperatures. PAH concentrations increased in relation to the number of rings in the fragment, with the high-ring (4-,5- and 6-ring) PAHs accounting for 89.70-92.30% and 99.78-99.80% of the total concentration and total toxic equivalent of PAHs. In addition, particulate-phase PAHs (0.1-1.0 µm), developed through the synergistic effect of PAHs and fine particles, were difficult to collect by fast condensation. Inorganic fine particles could be formed when ammonia-rich conditions prevail, reducing PAH condensation further. Furthermore, CPM was morphologically and chemically characterized. During the experiment, fine and well-aggregated CPMs were detected on the membrane, and the diameter of CPMs was further enhanced by the addition of 16 PAHs. Most of the C element was collected in the rinse fluid, thus indicating that PAHs in CPM were collected through condensation. Based on these findings, basic guidelines can be provided for the control of PAHs in flue gas from coal-fired power plants.

Novel biomarkers identifying hypertrophic cardiomyopathy and its obstructive variant based on targeted amino acid metabolomics.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is an underdiagnosed genetic heart disease worldwide. The management and prognosis of obstructive HCM (HOCM) and non-obstructive HCM (HNCM) are quite different, but it also remains challenging to discriminate these two subtypes. HCM is characterized by dysmetabolism, and myocardial amino acid (AA) metabolism is robustly changed. The present study aimed to delineate plasma AA and derivatives profiles, and identify potential biomarkers for HCM. METHODS: Plasma samples from 166 participants, including 57 cases of HOCM, 52 cases of HNCM, and 57 normal controls (NCs), who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020, were collected and analyzed by high-performance liquid chromatography-mass spectrometry based on targeted AA metabolomics. Three separate classification algorithms, including random forest, support vector machine, and logistic regression, were applied for the identification of specific AA and derivatives compositions for HCM and the development of screening models to discriminate HCM from NC as well as HOCM from HNCM. RESULTS: The univariate analysis showed that the serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels in the HCM group were significantly different from those in the NC group. Four AAs and derivatives (Panel A; proline, glycine, cysteine, and choline) were screened out by multiple feature selection algorithms for discriminating HCM patients from NCs. The receiver operating characteristic (ROC) analysis in Panel A yielded an area under the ROC curve (AUC) of 0.83 (0.75-0.91) in the training set and 0.79 (0.65-0.94) in the validation set. Moreover, among 10 AAs and derivatives (arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline) with statistical significance between HOCM and HNCM, 3 AAs (Panel B; arginine, proline, and ornithine) were selected to differentiate the two subgroups. The AUC values in the training and validation sets for Panel B were 0.83 (0.74-0.93) and 0.82 (0.66-0.98), respectively. CONCLUSIONS: The plasma AA and derivatives profiles were distinct between the HCM and NC groups. Based on the differential profiles, the two established screening models have potential value in assisting HCM screening and identifying whether it is obstructive.

LRRC8A critically regulates myofibroblast phenotypes and fibrotic remodeling following myocardial infarction.

Rationale: The transformation of fibroblasts into activated myofibroblasts is a critical step that results in cardiac fibrosis upon myocardial infarction (MI). Leucine-rich repeat-containing protein-8A (LRRC8A) is a multi-functional protein involved in cell survival, growth, and proliferation, whereas its role in regulating myofibroblast phenotypes and myocardial fibrosis remains unknown. Methods: Conditional myofibroblast-specific Lrrc8a knockout mouse models were established by crossing the Lrrc8aflox/flox mice with the tamoxifen-inducible periostin-Cre transgenic mice. The involvement of LRRC8A in regulating cardiac fibrosis post-MI and myofibroblast phenotypes induced by transforming growth factor-ß1 (TGF-ß1) was comprehensively evaluated. The mechanisms underlying LRRC8A regulation of myofibroblast phenotypes were determined by RNA sequencing-driven analysis followed by cause-effect experiments. Results: LRRC8A expression was significantly elevated in the fibrotic tissues and the fibroblasts isolated from the post-MI hearts. Compared with the wild-type (WT) littermates, the specific knockout of LRRC8A in myofibroblasts greatly attenuated myofibroblast transformation, fibrotic remodeling, and ventricular dysfunction after MI. Silencing of LRRC8A expression suppressed, whereas overexpression of LRRC8A enhanced, the pro-fibrotic myofibroblast phenotypes in isolated cardiac fibroblasts upon stimulation with TGF-ß1. LRRC8A participated in TGF-ß1-induced myofibroblast transformation via activating JAK2-STAT3 signaling. Furthermore, LRRC8A activated the JAK2-STAT3 pathway via its C-terminal leucine-rich repeat-domain (LRRD), directly interacting with growth factor receptor-bound protein 2 (GRB2), an adaptor protein associated with and necessary for tyrosine-phosphorylated JAK2. Conclusions: LRRC8A regulates myofibroblast transformation and cardiac fibrosis following MI. LRRC8A inhibition might be a promising strategy for cardiac fibrosis and heart failure.

Mesenchymal Stromal Cells Overexpressing Farnesoid X Receptor Exert Cardioprotective Effects Against Acute Ischemic Heart Injury by Binding Endogenous Bile Acids.

Bile acid metabolites have been increasingly recognized as pleiotropic signaling molecules that regulate cardiovascular functions, but their role in mesenchymal stromal cells (MSC)-based therapy has never been investigated. It is found that overexpression of farnesoid X receptor (FXR), a main receptor for bile acids, improves the retention and cardioprotection of adipose tissue-derived MSC (ADSC) administered by intramyocardial injection in mice with myocardial infarction (MI), which shows enhanced antiapoptotic, proangiogenic, and antifibrotic effects. RNA sequencing, LC-MS/MS, and loss-of-function studies reveal that FXR overexpression promotes ADSC paracrine angiogenesis via Angptl4. FXR overexpression improves ADSC survival in vivo but fails in vitro. By performing bile acid-targeted metabolomics using ischemic heart tissue, 19 bile acids are identified. Among them, cholic acid and deoxycholic acid significantly increase Angptl4 secretion from ADSC overexpressing FXR and further improve their proangiogenic capability. Moreover, ADSC overexpressing FXR shows significantly lower apoptosis by upregulating Nqo-1 expression only in the presence of FXR ligands. Retinoid X receptor α is identified as a coactivator of FXR. It is first demonstrated that there is a bile acid pool in the myocardial microenvironment. Targeting the bile acid-FXR axis may be a novel strategy for improving the curative effect of MSC-based therapy for MI.

Beclin1 haploinsufficiency compromises mesenchymal stem cell-offered cardioprotection against myocardial infarction.

Mesenchymal stem cells (MSCs)-based therapy has displayed some promises in ischemia heart diseases although its efficacy may be affected by changes in surrounding environments. This study evaluated the role of autophagy insufficiency using Beclin1 haploinsufficiency (BECN+/-) on intra-myocardial MSC transplantation-evoked effect against myocardial infarction. Donor MSCs from C57BL/6 mice were labelled with cell-tracker CM Dil and were delivered into LV free wall adjacent to infarct region in wild-type (WT) and BECN+/- recipient mice following ligation of left main coronary artery (MI-MSCs). Ten days following MI, myocardial function was assessed using echocardiography. Cardiomyocyte contractility and intracellular Ca2+ were monitored using cardiomyocytes from the area-at-risk adjacent to infarct. CM-Dil labeled cells were tracked in MSCs recipient mice using fluorescence microscopy. Lectin, Masson trichrome staining and Western blot analysis were employed to determine cardiomyocyte area, scar fibrosis, apoptosis and inflammation. MI insult triggered scar fibrosis, LV chamber dilation, decreased fractional shortening, ejection fraction, cardiomyocyte shortening, maximal velocity of shortening and relengthening as well as prolonged relengthening, which were abrogated or attenuated by MSCs therapy in WT but not BECN+/- mice. MI decreased intracellular Ca2+ rise and decay in response to electrical stimuli without affecting resting intracellular Ca2+, which were reconciled by MSCs in WT but not BECN+/- mice. MSCs further attenuated MI-induced mitochondrial ultrastructural injury, apoptosis, inflammation and autophagy defects in peri-infarct area in WT but not BECN+/- mice. Collectively, our results suggested that autophagy insufficiency dampened in MSCs-elicited cardioprotection associated with dampened apoptosis and inflammation.

Derivation and Validation of a Screening Model for Hypertrophic Cardiomyopathy Based on Electrocardiogram Features.

Background: Hypertrophic cardiomyopathy (HCM) is a widely distributed, but clinically heterogeneous genetic heart disease, affects approximately 20 million people worldwide. Nowadays, HCM is treatable with the advancement of medical interventions. However, due to occult clinical presentations and a lack of easy, inexpensive, and widely popularized screening approaches in the general population, 80-90% HCM patients are not clinically identifiable, which brings certain safety hazards could have been prevented. The majority HCM patients showed abnormal and diverse electrocardiogram (ECG) presentations, it is unclear which ECG parameters are the most efficient for HCM screening. Objective: We aimed to develop a pragmatic prediction model based on the most common ECG features to screen for HCM. Methods: Between April 1st and September 30th, 2020, 423 consecutive subjects from the International Cooperation Center for Hypertrophic Cardiomyopathy of Xijing Hospital [172 HCM patients, 251 participants without left ventricular hypertrophy (non-HCM)] were prospectively included in the training cohort. Between January 4th and February 30th, 2021, 163 participants from the same center were included in the temporal internal validation cohort (62 HCM patients, 101 non-HCM participants). External validation was performed using retrospectively collected ECG data from Xijing Hospital (3,232 HCM ECG samples from January 1st, 2000, to March 31st, 2020; 95,184 non-HCM ECG samples from January 1st to December 31st, 2020). The C-statistic was used to measure the discriminative ability of the model. Results: Among 30 ECG features examined, all except abnormal Q wave significantly differed between the HCM patients and non-HCM comparators. After several independent feature selection approaches and model evaluation, we included only two ECG features, T wave inversion (TWI) and the amplitude of S wave in lead V1 (SV1), in the HCM prediction model. The model showed a clearly useful discriminative performance (C-statistic > 0.75) in the training [C-statistic 0.857 (0.818-0.896)], and temporal validation cohorts [C-statistic 0.871 (0.812-0.930)]. In the external validation cohort, the C-statistic of the model was 0.833 [0.825-0.841]. A browser-based calculator was generated accordingly. Conclusion: The pragmatic model established using only TWI and SV1 may be helpful for predicting the probability of HCM and shows promise for use in population-based HCM screening.

Excessive branched-chain amino acid accumulation restricts mesenchymal stem cell-based therapy efficacy in myocardial infarction.

Mesenchymal stem cells (MSCs) delivered into the post-ischemic heart milieu have a low survival and retention rate, thus restricting the cardioreparative efficacy of MSC-based therapy. Chronic ischemia results in metabolic reprogramming in the heart, but little is known about how these metabolic changes influence implanted MSCs. Here, we found that excessive branched-chain amino acid (BCAA) accumulation, a metabolic signature seen in the post-ischemic heart, was disadvantageous to the retention and cardioprotection of intramyocardially injected MSCs. Discovery-driven experiments revealed that BCAA at pathological levels sensitized MSCs to stress-induced cell death and premature senescence via accelerating the loss of histone 3 lysine 9 trimethylation (H3K9me3). A novel mTORC1/DUX4/KDM4E axis was identified as the cause of BCAA-induced H3K9me3 loss and adverse phenotype acquisition. Enhancing BCAA catabolic capability in MSCs via genetic/pharmacological approaches greatly improved their adaptation to the high BCAA milieu and strengthened their cardioprotective efficacy. We conclude that aberrant BCAA accumulation is detrimental to implanted MSCs via a previously unknown metabolite-signaling-epigenetic mechanism, emphasizing that the metabolic changes of the post-ischemic heart crucially influence the fate of implanted MSCs and their therapeutic benefits.

Small Extracellular Vesicles From Brown Adipose Tissue Mediate Exercise Cardioprotection.

RATIONALE: Long-term exercise provides reliable cardioprotection via mechanisms still incompletely understood. Although traditionally considered a thermogenic tissue, brown adipose tissue (BAT) communicates with remote organs (eg, the heart) through its endocrine function. BAT expands in response to exercise, but its involvement in exercise cardioprotection remains undefined. OBJECTIVE: This study investigated whether small extracellular vesicles (sEVs) secreted by BAT and their contained microRNAs (miRNAs) regulate cardiomyocyte survival and participate in exercise cardioprotection in the context of myocardial ischemia/reperfusion (MI/R) injury. METHODS AND RESULTS: Four weeks of exercise resulted in a significant BAT expansion in mice. Surgical BAT ablation before MI/R weakened the salutary effects of exercise. Adeno-associated virus 9 vectors carrying short hairpin RNA targeting Rab27a (a GTPase required for sEV secretion) or control viruses were injected in situ into the interscapular BAT. Exercise-mediated protection against MI/R injury was greatly attenuated in mice whose BAT sEV secretion was suppressed by Rab27a silencing. Intramyocardial injection of the BAT sEVs ameliorated MI/R injury, revealing the cardioprotective potential of BAT sEVs. Discovery-driven experiments identified miR-125b-5p, miR-128-3p, and miR-30d-5p (referred to as the BAT miRNAs) as essential BAT sEV components for mediating cardioprotection. BAT-specific inhibition of the BAT miRNAs prevented their upregulation in plasma sEVs and hearts of exercised mice and attenuated exercise cardioprotection. Mechanistically, the BAT miRNAs cooperatively suppressed the proapoptotic MAPK (mitogen-associated protein kinase) pathway by targeting a series of molecules (eg, Map3k5, Map2k7, and Map2k4) in the signaling cascade. Delivery of BAT sEVs into hearts or cardiomyocytes suppressed MI/R-related MAPK pathway activation, an effect that disappeared with the combined use of the BAT miRNA inhibitors. CONCLUSIONS: The sEVs secreted by BAT participate in exercise cardioprotection via delivering the cardioprotective miRNAs into the heart. These results provide novel insights into the mechanisms underlying the BAT-cardiomyocyte interaction and highlight BAT sEVs and their contained miRNAs as alternative candidates for exercise cardioprotection.

Irisin Promotes Cardiac Homing of Intravenously Delivered MSCs and Protects against Ischemic Heart Injury.

Few intravenously administered mesenchymal stromal cells (MSCs) engraft to the injured myocardium, thereby limiting their therapeutic efficacy for the treatment of ischemic heart injury. Here, it is found that irisin pretreatment increases the cardiac homing of adipose tissue-derived MSCs (ADSCs) administered by single and multiple intravenous injections to mice with MI/R by more than fivefold, which subsequently increases their antiapoptotic, proangiogenic, and antifibrotic effects in rats and mice that underwent MI/R. RNA sequencing, Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis, and loss-of-function studies identified CSF2RB as a cytokine receptor that facilitates the chemotaxis of irisin-treated ADSCs in the presence of CSF2, a chemokine that is significantly upregulated in the ischemic heart. Cardiac-specific CSF2 knockdown blocked the cardiac homing and cardioprotection abilities of intravenously injected irisin-treated ADSCs in mice subjected to MI/R. Moreover, irisin pretreatment reduced the apoptosis of hydrogen peroxide-induced ADSCs and increased the paracrine proangiogenic effect of ADSCs. ERK1/2-SOD2, and ERK1/2-ANGPTL4 are responsible for the antiapoptotic and paracrine angiogenic effects of irisin-treated ADSCs, respectively. Integrin αV/ß5 is identified as the irisin receptor in ADSCs. These results provide compelling evidence that irisin pretreatment can be an effective means to optimize intravenously delivered MSCs as therapy for ischemic heart injury.

Targeting PPARα in low ambient temperature exposure-induced cardiac dysfunction and remodeling.

The present study demonstrates that the down-regulation of peroxisome proliferator-activated receptor-α (PPARα) results in chronic low ambient temperature (LT) exposure-induced cardiac dysfunction and remodeling, emphasizing the therapeutic potential of PPARα activation strategies (e.g. fenofibrate treatment) in LT-associated cardiac injury.

FNDC5/Irisin attenuates diabetic cardiomyopathy in a type 2 diabetes mouse model by activation of integrin αV/ß5-AKT signaling and reduction of oxidative/nitrosative stress.

Irisin, the cleaved form of the fibronectin type III domain containing 5 (FNDC5) protein, is involved in metabolism and inflammation. Recent findings indicated that irisin participated in cardiovascular physiology and pathology. In this study, we investigated the effects of FNDC5/irisin on diabetic cardiomyopathy (DCM) in type 2 diabetic db/db mice. Downregulation of myocardial FNDC5/irisin protein expression and plasma irisin levels was observed in db/db mice compared to db/+ controls. Moreover, echocardiography revealed that db/db mice exhibited normal cardiac systolic function and impaired diastolic function. Adverse structural remodeling, including cardiomyocyte apoptosis, myocardial fibrosis, and cardiac hypertrophy were observed in the hearts of db/db mice. Sixteen-week-old db/db mice were intramyocardially injected with adenovirus encoding FNDC5 or treated with recombinant human irisin via a peritoneal implant osmotic pump for 4 weeks. Both overexpression of myocardial FNDC5 and exogenous irisin administration attenuated diastolic dysfunction and cardiac structural remodeling in db/db mice. Results from in vitro studies revealed that FNDC5/irisin protein expression was decreased in high glucose (HG)/high fat (HF)-treated cardiomyocytes. Increased levels of inducible nitric oxide synthase (iNOS), NADPH oxidase 2 (NOX2), 3-nitrotyrosine (3-NT), reactive oxygen species (ROS), and peroxynitrite (ONOO-) in HG/HF-treated H9C2 cells provided evidence of oxidative/nitrosative stress, which was alleviated by treatment with FNDC5/irisin. Moreover, the mitochondria membrane potential (ΔΨm) was decreased and cytochrome C was released from mitochondria with increased levels of cleaved caspase-3 in HG/HF-treated H9C2 cells, indicating the presence of mitochondria-dependent apoptosis, which was partially reversed by FNDC5/irisin treatment. Mechanistic studies showed that activation of integrin αVß5-AKT signaling and attenuation of oxidative/nitrosative stress were responsible for the cardioprotective effects of FNDC5/irisin. Therefore, FNDC5/irisin mediates cardioprotection in DCM by inhibiting myocardial apoptosis, myocardial fibrosis, and cardiac hypertrophy. These findings implicate that FNDC5/irisin as a potential therapeutic intervention for DCM, especially in type 2 diabetes mellitus (T2DM).