Distinct transcription factor networks control neutrophil-driven inflammation.

Neutrophils display distinct gene expression patters depending on their developmental stage, activation state and tissue microenvironment. To determine the transcription factor networks that shape these responses in a mouse model, we integrated transcriptional and chromatin analyses of neutrophils during acute inflammation. We showed active chromatin remodeling at two transition stages: bone marrow-to-blood and blood-to-tissue. Analysis of differentially accessible regions revealed distinct sets of putative transcription factors associated with control of neutrophil inflammatory responses. Using ex vivo and in vivo approaches, we confirmed that RUNX1 and KLF6 modulate neutrophil maturation, whereas RELB, IRF5 and JUNB drive neutrophil effector responses and RFX2 and RELB promote survival. Interfering with neutrophil activation by targeting one of these factors, JUNB, reduced pathological inflammation in a mouse model of myocardial infarction. Therefore, our study represents a blueprint for transcriptional control of neutrophil responses in acute inflammation and opens possibilities for stage-specific therapeutic modulation of neutrophil function in disease.

Regulation of Cell Cycle to Stimulate Adult Cardiomyocyte Proliferation and Cardiac Regeneration.

Human diseases are often caused by loss of somatic cells that are incapable of re-entering the cell cycle for regenerative repair. Here, we report a combination of cell-cycle regulators that induce stable cytokinesis in adult post-mitotic cells. We screened cell-cycle regulators expressed in proliferating fetal cardiomyocytes and found that overexpression of cyclin-dependent kinase 1 (CDK1), CDK4, cyclin B1, and cyclin D1 efficiently induced cell division in post-mitotic mouse, rat, and human cardiomyocytes. Overexpression of the cell-cycle regulators was self-limiting through proteasome-mediated degradation of the protein products. In vivo lineage tracing revealed that 15%-20% of adult cardiomyocytes expressing the four factors underwent stable cell division, with significant improvement in cardiac function after acute or subacute myocardial infarction. Chemical inhibition of Tgf-ß and Wee1 made CDK1 and cyclin B dispensable. These findings reveal a discrete combination of genes that can efficiently unlock the proliferative potential in cells that have terminally exited the cell cycle.

Mesenchymal stromal cells for improvement of cardiac function following acute myocardial infarction: a matter of timing.

Acute myocardial infarction (AMI) is the leading cause of cardiovascular death and remains the most common cause of heart failure. Reopening of the occluded artery, i.e., reperfusion, is the only way to save the myocardium. However, the expected benefits of reducing infarct size are disappointing due to the reperfusion paradox, which also induces specific cell death. These ischemia-reperfusion (I/R) lesions can account for up to 50% of final infarct size, a major determinant for both mortality and the risk of heart failure (morbidity). In this review, we provide a detailed description of the cell death and inflammation mechanisms as features of I/R injury and cardioprotective strategies such as ischemic postconditioning as well as their underlying mechanisms. Due to their biological properties, the use of mesenchymal stromal/stem cells (MSCs) has been considered a potential therapeutic approach in AMI. Despite promising results and evidence of safety in preclinical studies using MSCs, the effects reported in clinical trials are not conclusive and even inconsistent. These discrepancies were attributed to many parameters such as donor age, in vitro culture, and storage time as well as injection time window after AMI, which alter MSC therapeutic properties. In the context of AMI, future directions will be to generate MSCs with enhanced properties to limit cell death in myocardial tissue and thereby reduce infarct size and improve the healing phase to increase postinfarct myocardial performance.

Spatial multi-omic map of human myocardial infarction.

Myocardial infarction is a leading cause of death worldwide1. Although advances have been made in acute treatment, an incomplete understanding of remodelling processes has limited the effectiveness of therapies to reduce late-stage mortality2. Here we generate an integrative high-resolution map of human cardiac remodelling after myocardial infarction using single-cell gene expression, chromatin accessibility and spatial transcriptomic profiling of multiple physiological zones at distinct time points in myocardium from patients with myocardial infarction and controls. Multi-modal data integration enabled us to evaluate cardiac cell-type compositions at increased resolution, yielding insights into changes of the cardiac transcriptome and epigenome through the identification of distinct tissue structures of injury, repair and remodelling. We identified and validated disease-specific cardiac cell states of major cell types and analysed them in their spatial context, evaluating their dependency on other cell types. Our data elucidate the molecular principles of human myocardial tissue organization, recapitulating a gradual cardiomyocyte and myeloid continuum following ischaemic injury. In sum, our study provides an integrative molecular map of human myocardial infarction, represents an essential reference for the field and paves the way for advanced mechanistic and therapeutic studies of cardiac disease.

Repair of the Infarcted Heart: Cellular Effectors, Molecular Mechanisms and Therapeutic Opportunities.

The adult mammalian heart has limited endogenous regenerative capacity and heals through the activation of inflammatory and fibrogenic cascades that ultimately result in the formation of a scar. After infarction, massive cardiomyocyte death releases a broad range of damage-associated molecular patterns that initiate both myocardial and systemic inflammatory responses. TLRs (toll-like receptors) and NLRs (NOD-like receptors) recognize damage-associated molecular patterns (DAMPs) and transduce downstream proinflammatory signals, leading to upregulation of cytokines (such as interleukin-1, TNF-α [tumor necrosis factor-α], and interleukin-6) and chemokines (such as CCL2 [CC chemokine ligand 2]) and recruitment of neutrophils, monocytes, and lymphocytes. Expansion and diversification of cardiac macrophages in the infarcted heart play a major role in the clearance of the infarct from dead cells and the subsequent stimulation of reparative pathways. Efferocytosis triggers the induction and release of anti-inflammatory mediators that restrain the inflammatory reaction and set the stage for the activation of reparative fibroblasts and vascular cells. Growth factor-mediated pathways, neurohumoral cascades, and matricellular proteins deposited in the provisional matrix stimulate fibroblast activation and proliferation and myofibroblast conversion. Deposition of a well-organized collagen-based extracellular matrix network protects the heart from catastrophic rupture and attenuates ventricular dilation. Scar maturation requires stimulation of endogenous signals that inhibit fibroblast activity and prevent excessive fibrosis. Moreover, in the mature scar, infarct neovessels acquire a mural cell coat that contributes to the stabilization of the microvascular network. Excessive, prolonged, or dysregulated inflammatory or fibrogenic cascades accentuate adverse remodeling and dysfunction. Moreover, inflammatory leukocytes and fibroblasts can contribute to arrhythmogenesis. Inflammatory and fibrogenic pathways may be promising therapeutic targets to attenuate heart failure progression and inhibit arrhythmia generation in patients surviving myocardial infarction.

Lymphoangiocrine signals promote cardiac growth and repair.

Recent studies have suggested that lymphatics help to restore heart function after cardiac injury1-6. Here we report that lymphatics promote cardiac growth, repair and cardioprotection in mice. We show that a lymphoangiocrine signal produced by lymphatic endothelial cells (LECs) controls the proliferation and survival of cardiomyocytes during heart development, improves neonatal cardiac regeneration and is cardioprotective after myocardial infarction. Embryos that lack LECs develop smaller hearts as a consequence of reduced cardiomyocyte proliferation and increased cardiomyocyte apoptosis. Culturing primary mouse cardiomyocytes in LEC-conditioned medium increases cardiomyocyte proliferation and survival, which indicates that LECs produce lymphoangiocrine signals that control cardiomyocyte homeostasis. Characterization of the LEC secretome identified the extracellular protein reelin (RELN) as a key component of this process. Moreover, we report that LEC-specific Reln-null mouse embryos develop smaller hearts, that RELN is required for efficient heart repair and function after neonatal myocardial infarction, and that cardiac delivery of RELN using collagen patches improves heart function in adult mice after myocardial infarction by a cardioprotective effect. These results highlight a lymphoangiocrine role of LECs during cardiac development and injury response, and identify RELN as an important mediator of this function.

Cardiac regenerative capacity and mechanisms.

The heart holds the monumental yet monotonous task of maintaining circulation. Although cardiac function is critical to other organs and to life itself, mammals are not equipped with significant natural capacity to replace heart muscle that has been lost by injury. This deficiency plays a role in leaving millions worldwide vulnerable to heart failure each year. By contrast, certain other vertebrate species such as zebrafish are strikingly good at heart regeneration. A cellular and molecular understanding of endogenous regenerative mechanisms and advances in methodology to transplant cells together project a future in which cardiac muscle regeneration can be therapeutically stimulated in injured human hearts. This review focuses on what has been discovered recently about cardiac regenerative capacity and how natural mechanisms of heart regeneration in model systems are stimulated and maintained.

Targeting NPM1 Epigenetically Promotes Postinfarction Cardiac Repair by Reprogramming Reparative Macrophage Metabolism.

BACKGROUND: Reparative macrophages play a crucial role in limiting excessive fibrosis and promoting cardiac repair after myocardial infarction (MI), highlighting the significance of enhancing their reparative phenotype for wound healing. Metabolic adaptation orchestrates the phenotypic transition of macrophages; however, the precise mechanisms governing metabolic reprogramming of cardiac reparative macrophages remain poorly understood. In this study, we investigated the role of NPM1 (nucleophosmin 1) in the metabolic and phenotypic shift of cardiac macrophages in the context of MI and explored the therapeutic effect of targeting NPM1 for ischemic tissue repair. METHODS: Peripheral blood mononuclear cells were obtained from healthy individuals and patients with MI to explore NPM1 expression and its correlation with prognostic indicators. Through RNA sequencing, metabolite profiling, histology, and phenotype analyses, we investigated the role of NPM1 in postinfarct cardiac repair using macrophage-specific NPM1 knockout mice. Epigenetic experiments were conducted to study the mechanisms underlying metabolic reprogramming and phenotype transition of NPM1-deficient cardiac macrophages. The therapeutic efficacy of antisense oligonucleotide and inhibitor targeting NPM1 was then assessed in wild-type mice with MI. RESULTS: NPM1 expression was upregulated in the peripheral blood mononuclear cells from patients with MI that closely correlated with adverse prognostic indicators of MI. Macrophage-specific NPM1 deletion reduced infarct size, promoted angiogenesis, and suppressed tissue fibrosis, in turn improving cardiac function and protecting against adverse cardiac remodeling after MI. Furthermore, NPM1 deficiency boosted the reparative function of cardiac macrophages by shifting macrophage metabolism from the inflammatory glycolytic system to oxygen-driven mitochondrial energy production. The oligomeric NPM1 recruited histone demethylase KDM5b to the promoter of Tsc1 (TSC complex subunit 1), the mTOR (mechanistic target of rapamycin kinase) complex inhibitor, reduced histone H3K4me3 modification, and inhibited TSC1 expression, which then facilitated mTOR-related inflammatory glycolysis and antagonized the reparative function of cardiac macrophages. The in vivo administration of antisense oligonucleotide targeting NPM1 or oligomerization inhibitor NSC348884 substantially ameliorated tissue injury and enhanced cardiac recovery in mice after MI. CONCLUSIONS: Our findings uncover the key role of epigenetic factor NPM1 in impeding postinfarction cardiac repair by remodeling metabolism pattern and impairing the reparative function of cardiac macrophages. NPM1 may serve as a promising prognostic biomarker and a valuable therapeutic target for heart failure after MI.

ABCG2-Expressing Clonal Repopulating Endothelial Cells Serve to Form and Maintain Blood Vessels.

BACKGROUND: Most organs are maintained lifelong by resident stem/progenitor cells. During development and regeneration, lineage-specific stem/progenitor cells can contribute to the growth or maintenance of different organs, whereas fully differentiated mature cells have less regenerative potential. However, it is unclear whether vascular endothelial cells (ECs) are also replenished by stem/progenitor cells with EC-repopulating potential residing in blood vessels. It has been reported recently that some EC populations possess higher clonal proliferative potential and vessel-forming capacity compared with mature ECs. Nevertheless, a marker to identify vascular clonal repopulating ECs (CRECs) in murine and human individuals is lacking, and, hence, the mechanism for the proliferative, self-renewal, and vessel-forming potential of CRECs is elusive. METHODS: We analyzed colony-forming, self-renewal, and vessel-forming potential of ABCG2 (ATP binding cassette subfamily G member 2)-expressing ECs in human umbilical vessels. To study the contribution of Abcg2-expressing ECs to vessel development and regeneration, we developed Abcg2CreErt2;ROSA TdTomato mice and performed lineage tracing during mouse development and during tissue regeneration after myocardial infarction injury. RNA sequencing and chromatin methylation chromatin immunoprecipitation followed by sequencing were conducted to study the gene regulation in Abcg2-expressing ECs. RESULTS: In human and mouse vessels, ECs with higher ABCG2 expression (ABCECs) possess higher clonal proliferative potential and in vivo vessel-forming potential compared with mature ECs. These cells could clonally contribute to vessel formation in primary and secondary recipients after transplantation. These features of ABCECs meet the criteria of CRECs. Results from lineage tracing experiments confirm that Abcg2-expressing CRECs (AbcCRECs) contribute to arteries, veins, and capillaries in cardiac tissue development and vascular tissue regeneration after myocardial infarction. Transcriptome and epigenetic analyses reveal that a gene expression signature involved in angiogenesis and vessel development is enriched in AbcCRECs. In addition, various angiogenic genes, such as Notch2 and Hey2, are bivalently modified by trimethylation at the 4th and 27th lysine residue of histone H3 (H3K4me3 and H3K27me3) in AbcCRECs. CONCLUSIONS: These results are the first to establish that a single prospective marker identifies CRECs in mice and human individuals, which holds promise to provide new cell therapies for repair of damaged vessels in patients with endothelial dysfunction.

Piezo1-Mediated Neurogenic Inflammatory Cascade Exacerbates Ventricular Remodeling After Myocardial Infarction.

BACKGROUND: Heart failure is associated with a high rate of mortality and morbidity, and ventricular remodeling invariably precedes heart failure. Ventricular remodeling is fundamentally driven by mechanotransduction that is regulated by both the nervous system and the immune system. However, it remains unknown which key molecular factors govern the neuro/immune/cardio axis that underlies mechanotransduction during ventricular remodeling. Here, we investigated whether the mechanosensitive Piezo cation channel-mediated neurogenic inflammatory cascade underlies ventricular remodeling-related mechanotransduction. METHODS: By ligating the left coronary artery of rats to establish an in vivo model of chronic myocardial infarction (MI), lentivirus-mediated thoracic dorsal root ganglion (TDRG)-specific Piezo1 knockdown rats and adeno-associated virus-PHP.S-mediated TDRG neuron-specific Piezo1 knockout mice were used to investigate whether Piezo1 in the TDRG plays a functional role during ventricular remodeling. Subsequently, neutralizing antibody-mediated TDRG IL-6 (interleukin-6) inhibition rats and adeno-associated virus-PHP.S-mediated TDRG neuron-specific IL-6 knockdown mice were used to determine the mechanism underlying neurogenic inflammation. Primary TDRG neurons were used to evaluate Piezo1 function in vitro. RESULTS: Expression of Piezo1 and IL-6 was increased, and these factors were functionally activated in TDRG neurons at 4 weeks after MI. Both knockdown of TDRG-specific Piezo1 and deletion of TDRG neuron-specific Piezo1 lessened the severity of ventricular remodeling at 4 weeks after MI and decreased the level of IL-6 in the TDRG or heart. Furthermore, inhibition of TDRG IL-6 or knockdown of TDRG neuron-specific IL-6 also ameliorated ventricular remodeling and suppressed the IL-6 cascade in the heart, whereas the Piezo1 level in the TDRG was not affected. In addition, enhanced Piezo1 function, as reflected by abundant calcium influx induced by Yoda1 (a selective agonist of Piezo1), led to increased release of IL-6 from TDRG neurons in mice 4 weeks after MI. CONCLUSIONS: Our findings point to a critical role for Piezo1 in ventricular remodeling at 4 weeks after MI and reveal a neurogenic inflammatory cascade as a previously unknown facet of the neuronal immune signaling axis underlying mechanotransduction.

Small Extracellular Vesicles From Infarcted and Failing Heart Accelerate Tumor Growth.

BACKGROUND: Myocardial infarction (MI) and heart failure are associated with an increased incidence of cancer. However, the mechanism is complex and unclear. Here, we aimed to test our hypothesis that cardiac small extracellular vesicles (sEVs), particularly cardiac mesenchymal stromal cell-derived sEVs (cMSC-sEVs), contribute to the link between post-MI left ventricular dysfunction (LVD) and cancer. METHODS: We purified and characterized sEVs from post-MI hearts and cultured cMSCs. Then, we analyzed cMSC-EV cargo and proneoplastic effects on several lines of cancer cells, macrophages, and endothelial cells. Next, we modeled heterotopic and orthotopic lung and breast cancer tumors in mice with post-MI LVD. We transferred cMSC-sEVs to assess sEV biodistribution and its effect on tumor growth. Finally, we tested the effects of sEV depletion and spironolactone treatment on cMSC-EV release and tumor growth. RESULTS: Post-MI hearts, particularly cMSCs, produced more sEVs with proneoplastic cargo than nonfailing hearts did. Proteomic analysis revealed unique protein profiles and higher quantities of tumor-promoting cytokines, proteins, and microRNAs in cMSC-sEVs from post-MI hearts. The proneoplastic effects of cMSC-sEVs varied with different types of cancer, with lung and colon cancers being more affected than melanoma and breast cancer cell lines. Post-MI cMSC-sEVs also activated resting macrophages into proangiogenic and protumorigenic states in vitro. At 28-day follow-up, mice with post-MI LVD developed larger heterotopic and orthotopic lung tumors than did sham-MI mice. Adoptive transfer of cMSC-sEVs from post-MI hearts accelerated the growth of heterotopic and orthotopic lung tumors, and biodistribution analysis revealed accumulating cMSC-sEVs in tumor cells along with accelerated tumor cell proliferation. sEV depletion reduced the tumor-promoting effects of MI, and adoptive transfer of cMSC-sEVs from post-MI hearts partially restored these effects. Finally, spironolactone treatment reduced the number of cMSC-sEVs and suppressed tumor growth during post-MI LVD. CONCLUSIONS: Cardiac sEVs, specifically cMSC-sEVs from post-MI hearts, carry multiple protumorigenic factors. Uptake of cMSC-sEVs by cancer cells accelerates tumor growth. Treatment with spironolactone significantly reduces accelerated tumor growth after MI. Our results provide new insight into the mechanism connecting post-MI LVD to cancer and propose a translational option to mitigate this deadly association.

Uterine Immunoprivileged Cells Restore Cardiac Function of Male Recipients After Myocardial Infarction.

It has been documented that the uterus plays a key cardio-protective role in pre-menopausal women, which is supported by uterine cell therapy, to preserve cardiac functioning post-myocardial infarction, being effective among females. However, whether such therapies would also be beneficial among males is still largely unknown. In this study, we aimed to fill in this gap in knowledge by examining the effects of transplanted uterine cells on infarcted male hearts. We identified, based on major histocompatibility complex class I (MHC-I) expression levels, 3 uterine reparative cell populations: MHC-I(neg), MHC-I(mix), and MHC-I(pos). In vitro, MHC-I(neg) cells showed higher levels of pro-angiogenic, pro-survival, and anti-inflammatory factors, compared to MHC-I(mix) and MHC-I(pos). Furthermore, when cocultured with allogeneic mixed leukocytes, MHC-I(neg) had lower cytotoxicity and leukocyte proliferation. In particular, CD8+ cytotoxic T cells significantly decreased, while CD4+CD25+ Tregs and CD4-CD8- double-negative T cells significantly increased when cocultured with MHC-I(neg), compared to MHC-I(mix) and MHC-I(pos) cocultures. In vivo, MHC-I(neg) as well as MHC-I(mix) were found under both syngeneic and allogeneic transplantation in infarcted male hearts, to significantly improve cardiac function and reduce the scar size, via promoting angiogenesis in the infarcted area. All of these findings thus support the view that males could also benefit from the cardio-protective effects observed among females, via cell therapy approaches involving the transplantation of immuno-privileged uterine reparative cells in infarcted hearts.

Aerobic training attenuates cardiac remodeling in mice post-myocardial infarction by inhibiting the p300/CBP-associated factor.

Aerobic training (AT), an effective form of cardiac rehabilitation, has been shown to be beneficial for cardiac repair and remodeling after myocardial infarction (MI). The p300/CBP-associated factor (PCAF) is one of the most important lysine acetyltransferases and is involved in various biological processes. However, the role of PCAF in AT and AT-mediated cardiac remodeling post-MI has not been determined. Here, we found that the PCAF protein level was significantly increased after MI, while AT blocked the increase in PCAF. AT markedly improved cardiac remodeling in mice after MI by reducing endoplasmic reticulum stress (ERS). In vivo, similar to AT, pharmacological inhibition of PCAF by Embelin improved cardiac recovery and attenuated ERS in MI mice. Furthermore, we observed that both IGF-1, a simulated exercise environment, and Embelin protected from H2O2-induced cardiomyocyte injury, while PCAF overexpression by viruses or the sirtuin inhibitor nicotinamide eliminated the protective effect of IGF-1 in H9C2 cells. Thus, our data indicate that maintaining low PCAF levels plays an essential role in AT-mediated cardiac protection, and PCAF inhibition represents a promising therapeutic target for attenuating cardiac remodeling after MI.

The detrimental role of galectin-3 and endoplasmic reticulum stress in the cardiac consequences of myocardial ischemia in the context of obesity.

The association between cardiac fibrosis and galectin-3 was evaluated in patients with acute myocardial infarction (MI). The role of galectin-3 and its association with endoplasmic reticulum (ER) stress activation in the progression of cardiovascular fibrosis was also evaluated in obese-infarcted rats. The inhibitor of galectin-3 activity, modified citrus pectin (MCP; 100 mg/kg/day), and the inhibitor of the ER stress activation, 4-phenylbutyric acid (4-PBA; 500 mg/kg/day), were administered for 4 weeks after MI in obese rats. Overweight-obese patients who suffered a first MI showed higher circulating galectin-3 levels, higher extracellular volume, and LV infarcted size, as well as lower E/e'ratio and LVEF compared with normal-weight patients. A correlation was observed between galectin-3 levels and extracellular volume. Obese-infarcted animals presented cardiac hypertrophy and reduction in LVEF, and E/A ratio as compared with control animals. They also showed an increase in galectin-3 gene expression, as well as cardiac fibrosis and reduced autophagic flux. These alterations were associated with ER stress activation characterized by enhanced cardiac levels of binding immunoglobulin protein, which were correlated with those of galectin-3. Both MCP and 4-PBA not only reduced cardiac fibrosis, oxidative stress, galectin-3 levels, and ER stress activation, but also prevented cardiac functional alterations and ameliorated autophagic flux. These results show the relevant role of galectin-3 in the development of diffuse fibrosis associated with MI in the context of obesity in both the animal model and patients. Galectin-3 in tandem with ER stress activation could modulate different downstream mechanisms, including inflammation, oxidative stress, and autophagy.

Inhibition of Fap Promotes Cardiac Repair by Stabilizing BNP.

BACKGROUND: Myocardial infarction (MI) elicits cardiac fibroblast activation and extracellular matrix (ECM) deposition to maintain the structural integrity of the heart. Recent studies demonstrate that Fap (fibroblast activation protein)-a prolyl-specific serine protease-is an important marker of activated cardiac fibroblasts after MI. METHODS: Left ventricle and plasma samples from patients and healthy donors were used to analyze the expression level of FAP and its prognostic value. Echocardiography and histological analysis of heart sections were used to analyze cardiac functions, scar formation, ECM deposition and angiogenesis after MI. RNA-Sequencing, biochemical analysis, cardiac fibroblasts (CFs) and endothelial cells co-culture were used to reveal the molecular and cellular mechanisms by which Fap regulates angiogenesis. RESULTS: We found that Fap is upregulated in patient cardiac fibroblasts after cardiac injuries, while plasma Fap is downregulated and functions as a prognostic marker for cardiac repair. Genetic or pharmacological inhibition of Fap in mice significantly improved cardiac function after MI. Histological and transcriptomic analyses showed that Fap inhibition leads to increased angiogenesis in the peri-infarct zone, which promotes ECM deposition and alignment by cardiac fibroblasts and prevents their overactivation, thereby limiting scar expansion. Mechanistically, we found that BNP (brain natriuretic peptide) is a novel substrate of Fap that mediates postischemic angiogenesis. Fap degrades BNP to inhibit vascular endothelial cell migration and tube formation. Pharmacological inhibition of Fap in Nppb (encoding pre-proBNP) or Npr1 (encoding the BNP receptor)-deficient mice showed no cardioprotective effects, suggesting that BNP is a physiological substrate of Fap. CONCLUSIONS: This study identifies Fap as a negative regulator of cardiac repair and a potential drug target to treat MI. Inhibition of Fap stabilizes BNP to promote angiogenesis and cardiac repair.

Platelet-Derived MicroRNAs Regulate Cardiac Remodeling After Myocardial Ischemia.

BACKGROUND: Platelets can infiltrate ischemic myocardium and are increasingly recognized as critical regulators of inflammatory processes during myocardial ischemia and reperfusion (I/R). Platelets contain a broad repertoire of microRNAs (miRNAs), which, under certain conditions such as myocardial ischemia, may be transferred to surrounding cells or released into the microenvironment. Recent studies could demonstrate that platelets contribute substantially to the circulating miRNA pool holding the potential for so far undiscovered regulatory functions. The present study aimed to determine the role of platelet-derived miRNAs in myocardial injury and repair following myocardial I/R. METHODS: In vivo model of myocardial I/R, multimodal in vivo and ex vivo imaging approaches (light-sheet fluorescence microscopy, positron emission tomography and magnetic resonance imaging, speckle-tracking echocardiography) of myocardial inflammation and remodeling, and next-generation deep sequencing analysis of platelet miRNA expression. RESULTS: In mice with a megakaryocyte/platelet-specific knockout of pre-miRNA processing ribonuclease Dicer, the present study discloses a key role of platelet-derived miRNAs in the tightly regulated cellular processes orchestrating left ventricular remodeling after myocardial I/R following transient left coronary artery ligation. Disruption of the miRNA processing machinery in platelets by deletion of Dicer resulted in increased myocardial inflammation, impaired angiogenesis, and accelerated development of cardiac fibrosis, culminating in an increased infarct size by d7 that persisted through d28 of myocardial I/R. Worsened cardiac remodeling after myocardial infarction in mice with a platelet-specific Dicer deletion resulted in an increased fibrotic scar formation and distinguishably increased perfusion defect of the apical and anterolateral wall at day 28 post-myocardial infarction. Altogether, these observations culminated in an impaired left ventricular function and hampered long-term cardiac recovery after experimental myocardial infarction and reperfusion therapy. Treatment with the P2Y12 (P2Y purinoceptor 12) antagonist ticagrelor completely reversed increased myocardial damage and adverse cardiac remodeling observed in DicerPf4∆/Pf4∆ mice. CONCLUSIONS: The present study discloses a critical role of platelet-derived miRNA in myocardial inflammation and structural remodeling processes following myocardial I/R.

ZBP1 Protects Against mtDNA-Induced Myocardial Inflammation in Failing Hearts.

BACKGROUND: Mitochondrial DNA (mtDNA)-induced myocardial inflammation is intimately involved in cardiac remodeling. ZBP1 (Z-DNA binding protein 1) is a pattern recognition receptor positively regulating inflammation in response to mtDNA in inflammatory cells, fibroblasts, and endothelial cells. However, the role of ZBP1 in myocardial inflammation and cardiac remodeling remains unclear. The aim of this study was to elucidate the role of ZBP1 in mtDNA-induced inflammation in cardiomyocytes and failing hearts. METHODS: mtDNA was administrated into isolated cardiomyocytes. Myocardial infarctionwas conducted in wild type and ZBP1 knockout mice. RESULTS: We here found that, unlike in macrophages, ZBP1 knockdown unexpectedly exacerbated mtDNA-induced inflammation such as increases in IL (interleukin)-1ß and IL-6, accompanied by increases in RIPK3 (receptor interacting protein kinase 3), phosphorylated NF-κB (nuclear factor-κB), and NLRP3 (nucleotide-binding domain and leucine-rich-repeat family pyrin domain containing 3) in cardiomyocytes. RIPK3 knockdown canceled further increases in phosphorylated NF-κB, NLRP3, IL-1ß, and IL-6 by ZBP1 knockdown in cardiomyocytes in response to mtDNA. Furthermore, NF-κB knockdown suppressed such increases in NLRP3, IL-1ß, and IL-6 by ZBP1 knockdown in response to mtDNA. CpG-oligodeoxynucleotide, a Toll-like receptor 9 stimulator, increased RIPK3, IL-1ß, and IL-6 and ZBP1 knockdown exacerbated them. Dloop, a component of mtDNA, but not Tert and B2m, components of nuclear DNA, was increased in cytosolic fraction from noninfarcted region of mouse hearts after myocardial infarction compared with control hearts. Consistent with this change, ZBP1, RIPK3, phosphorylated NF-κB, NLRP3, IL-1ß, and IL-6 were increased in failing hearts. ZBP1 knockout mice exacerbated left ventricular dilatation and dysfunction after myocardial infarction, accompanied by further increases in RIPK3, phosphorylated NF-κB, NLRP3, IL-1ß, and IL-6. In histological analysis, ZBP1 knockout increased interstitial fibrosis and myocardial apoptosis in failing hearts. CONCLUSIONS: Our study reveals unexpected protective roles of ZBP1 against cardiac remodeling as an endogenous suppressor of mtDNA-induced myocardial inflammation.

Upregulation of P2X7 Exacerbates Myocardial Ischemia-Reperfusion Injury through Enhancing Inflammation and Apoptosis in Diabetic Mice.

Diabetes-aggravated myocardial ischemia-reperfusion (MI/R) injury remains an urgent medical issue, and the molecular mechanisms involved with diabetes and MI/R injury remain largely unknown. Previous studies have shown that inflammation and P2X7 signaling participate in the pathogenesis of the heart under individual conditions. It remains to be explored if P2X7 signaling is exacerbated or alleviated under double insults. We established a high-fat diet and streptozotocin-induced diabetic mouse model, and we compared the differences in immune cell infiltration and P2X7 expression between diabetic and nondiabetic mice after 24 h of reperfusion. The antagonist and agonist of P2X7 were administered before and after MI/R. Our study showed that the MI/R injury of diabetic mice was characterized by increased infarct area, impaired ventricular contractility, more apoptosis, aggravated immune cell infiltration, and overactive P2X7 signaling compared with nondiabetic mice. The major trigger of increased P2X7 was the MI/R-induced recruitment of monocytes and macrophages, and diabetes can be a synergistic factor in this process. Administration of P2X7 agonist eliminated the differences in MI/R injury between nondiabetic mice and diabetic mice. Both 2 wk of brilliant blue G injection before MI/R and acutely administered A438079 at the time of MI/R injury attenuated the role of diabetes in exacerbating MI/R injury, as evidenced by decreased infarct size, improved cardiac function, and inhibition of apoptosis. Additionally, brilliant blue G blockade decreased the heart rate after MI/R, which was accompanied by downregulation of tyrosine hydroxylase expression and nerve growth factor transcription. In conclusion, targeting P2X7 may be a promising strategy for reducing the risk of MI/R injury in diabetes.

Direct differentiation of rat skin fibroblasts into cardiomyocytes.

Myocardial infarction (MI) is one of the major cardiovascular diseases caused by diminished supply of nutrients and oxygen to the heart due to obstruction of the coronary artery. Different treatment options are available for cardiac diseases, however, they do not completely repair the damage. Therefore, reprogramming terminally differentiated fibroblasts using transcription factors is a promising strategy to differentiate them into cardiac like cells in vitro and to increase functional cardiomyocytes and reduce fibrotic scar in vivo. In this study, skin fibroblasts were selected for reprogramming because they serve as a convenient source for the autologous cell therapy. Fibroblasts were isolated from skin of rat pups, propagated, and directly reprogrammed towards cardiac lineage. For reprogramming, two different approaches were adopted, i.e., cells were transfected with: (1) combination of cardiac transcription factors; GATA4, MEF2c, Nkx2.5 (GMN), and (2) combination of cardiac transcription factors; GATA4, MEF2c, Nkx2.5, and iPSC factors; Oct4, Klf4, Sox2 and cMyc (GMNO). After 72 h of transfection, cells were analyzed for the expression of cardiac markers at the mRNA and protein levels. For in vivo study, rat MI models were developed by ligating the left anterior descending coronary artery and the reprogrammed cells were transplanted in the infarcted heart. qPCR results showed that the reprogrammed cells exhibited significant upregulation of cardiac genes. Immunocytochemistry analysis further confirmed cardiomyogenic differentiation of the reprogrammed cells. For the assessment of cardiac function, animals were analyzed via echocardiography after 2 and 4 weeks of cell transplantation. Echocardiographic results showed that the hearts transplanted with the reprogrammed cells improved ejection fraction, fractional shortening, left ventricular internal systolic and diastolic dimensions, and end systolic and diastolic volumes. After 4 weeks of cell transplantation, heart tissues were harvested and processed for histology. The histological analysis showed that the reprogrammed cells improved wall thickness of left ventricle and reduced fibrosis significantly as compared to the control. It is concluded from the study that novel combination of cardiac transcription factors directly reprogrammed skin fibroblasts and differentiated them into cardiomyocytes. These differentiated cells showed cardiomyogenic characters in vitro, and reduced fibrosis and improved cardiac function in vivo. Furthermore, direct reprogramming of fibroblasts transfected with cardiac transcription factors showed better regeneration of the injured myocardium and improved cardiac function as compared to the indirect approach in which combination of cardiac and iPSC factors were used. The study after further optimization could be used as a better strategy for cell-based therapeutic approaches for cardiovascular diseases.

KN-93 promotes HDAC4 nucleus translocation to promote fatty acid oxidation in myocardial infarction.

Myocardial infarction (MI) is a potentially fatal disease that causes a significant number of deaths worldwide. The strategy of increasing fatty acid oxidation in myocytes is considered a therapeutic avenue to accelerate metabolism to meet energy demands. We conducted the study aiming to investigate the effect of KN-93, which induces histone deacetylase (HDAC)4 shuttling to the nucleus, on fatty acid oxidation and the expression of related genes. A mouse model of myocardial infarction was induced by isoprenaline administration. Heart damage was assessed by the detection of cardiac injury markers. The level of fatty acid oxidation level was evaluated by testing the expression of related genes. Both immunofluorescence and immunoblotting in the cytosol or nucleus were utilized to observe the distribution of HDAC4. The interaction between HDAC4 and specificity protein (SP)1 was confirmed by co-immunoprecipitation. The acetylation level of SP1 was tested after KN-93 treatment and HDAC4 inhibitor. Oxygen consumption rate and immunoblotting experiments were used to determine whether the effect of KN-93 on increasing fatty acid oxidation is through HDAC4 and SP1. Administration of KN-93 significantly reduced cardiac injury in myocardial infarction and promoted fatty acid oxidation both in vitro and in vivo. KN-93 was shown to mediate nuclear translocation of HDAC4. HDAC4 was found to interact with SP1 and reduce SP1 acetylation. HDAC4 or SP1 inhibitors attenuated the effect of KN-93 on fatty acid oxidation. In conclusion, KN-93 promotes HDAC4 translocation to the nucleus, thereby potentially enhancing fatty acid oxidation by SP1.