hESC-Derived Epicardial Cells Promote Repair of Infarcted Hearts in Mouse and Swine.

Myocardial infarction (MI) causes excessive damage to the myocardium, including the epicardium. However, whether pluripotent stem cell-derived epicardial cells (EPs) can be a therapeutic approach for infarcted hearts remains unclear. Here, the authors report that intramyocardial injection of human embryonic stem cell-derived EPs (hEPs) at the acute phase of MI ameliorates functional worsening and scar formation in mouse hearts, concomitantly with enhanced cardiomyocyte survival, angiogenesis, and lymphangiogenesis. Mechanistically, hEPs suppress MI-induced infiltration and cytokine-release of inflammatory cells and promote reparative macrophage polarization. These effects are blocked by a type I interferon (IFN-I) receptor agonist RO8191. Moreover, intelectin 1 (ITLN1), abundantly secreted by hEPs, interacts with IFN-ß and mimics the effects of hEP-conditioned medium in suppression of IFN-ß-stimulated responses in macrophages and promotion of reparative macrophage polarization, whereas ITLN1 downregulation in hEPs cancels beneficial effects of hEPs in anti-inflammation, IFN-I response inhibition, and cardiac repair. Further, similar beneficial effects of hEPs are observed in a clinically relevant porcine model of reperfused MI, with no increases in the risk of hepatic, renal, and cardiac toxicity. Collectively, this study reveals hEPs as an inflammatory modulator in promoting infarct healing via a paracrine mechanism and provides a new therapeutic approach for infarcted hearts.

Small molecule QF84139 ameliorates cardiac hypertrophy via activating the AMPK signaling pathway.

Cardiac hypertrophy is a common adaptive response to a variety of stimuli, but prolonged hypertrophy leads to heart failure. Hence, discovery of agents treating cardiac hypertrophy is urgently needed. In the present study, we investigated the effects of QF84139, a newly synthesized pyrazine derivative, on cardiac hypertrophy and the underlying mechanisms. In neonatal rat cardiomyocytes (NRCMs), pretreatment with QF84139 (1-10 µM) concentration-dependently inhibited phenylephrine-induced hypertrophic responses characterized by fetal genes reactivation, increased ANP protein level and enlarged cardiomyocytes. In adult male mice, administration of QF84139 (5-90 mg·kg-1·d-1, i.p., for 2 weeks) dose-dependently reversed transverse aortic constriction (TAC)-induced cardiac hypertrophy displayed by cardiomyocyte size, left ventricular mass, heart weights, and reactivation of fetal genes. We further revealed that QF84139 selectively activated the AMPK signaling pathway without affecting the phosphorylation of CaMKIIδ, ERK1/2, AKT, PKCε, and P38 kinases in phenylephrine-treated NRCMs and in the hearts of TAC-treated mice. In NRCMs, QF84139 did not show additive effects with metformin on the AMPK activation, whereas the anti-hypertrophic effect of QF84139 was abolished by an AMPK inhibitor Compound C or knockdown of AMPKα2. In AMPKα2-deficient mice, the anti-hypertrophic effect of QF84139 was also vanished. These results demonstrate that QF84139 attenuates the PE- and TAC-induced cardiac hypertrophy via activating the AMPK signaling. This structurally novel compound would be a promising lead compound for developing effective agents for the treatment of cardiac hypertrophy.

Downregulation of LAPTM4B Contributes to the Impairment of the Autophagic Flux via Unopposed Activation of mTORC1 Signaling During Myocardial Ischemia/Reperfusion Injury.

RATIONALE: Impaired autophagic flux contributes to ischemia/reperfusion (I/R)-induced cardiomyocyte death, but the underlying molecular mechanisms remain largely unexplored. OBJECTIVE: To determine the role of LAPTM4B (lysosomal-associated transmembrane protein 4B) in the regulation of autophagic flux and myocardial I/R injury. METHODS AND RESULTS: LAPTM4B was expressed in murine hearts but downregulated in hearts with I/R (30 minutes/2 hours) injury and neonatal rat cardiomyocytes with hypoxia/reoxygenation (6 hours/2 hours) injury. During myocardial reperfusion, LAPTM4B-knockout (LAPTM4B-/-) mice had a significantly increased infarct size and lactate dehydrogenase release, whereas adenovirus-mediated LAPTM4B-overexpression was cardioprotective. Concomitantly, LAPTM4B-/- mice showed higher accumulation of the autophagy markers LC3-II (microtubule-associated protein 1A/1B-light chain 3), but not P62, in the I/R heart, whereas they did not alter chloroquine-induced further increases of LC3-II and P62 in both sham and I/R hearts. Conversely, LAPTM4B-overexpression had opposite effects. The hypoxia/reoxygenation-reduced viability of neonatal rat cardiomyocytes, ratio of autolysosomes/autophagosomes, and function of lysosomes were further decreased by LAPTM4B-knockdown but reversed by LAPTM4B-overexpression. Moreover, the LAPTM4B-overexpression-mediated benefits were abolished by knockdown of lysosome-associated membrane protein-2 (an autophagosome-lysosome fusion protein) in vivo and by the autophagy inhibitor bafilomycin A1 in vivo. In contrast, rapamycin (Rapa) successfully restored the impaired autophagic flux in LAPTM4B-/- mice and the subsequent myocardial I/R injury. Mechanistically, LAPTM4B regulated the activity of mTORC1 (mammalian target of rapamycin complex 1) via interacting with mTOR through its EC3 (extracelluar) domain. Thus, mTORC1 was overactivated in LAPTM4B-/- mice, leading to the repression of TFEB (transcription factor EB), a master regulator of lysosomal and autophagic genes, during myocardial I/R. The mTORC1 inhibition or TFEB-overexpression rescued the LAPTM4B-/--induced impairment in autophagic flux and I/R injury, whereas TFEB-knockdown abolished the LAPTM4B-overexpression-mediated recovery of autophagic flux and cardioprotection. CONCLUSIONS: The downregulation of LAPTM4B contributes to myocardial I/R-induced impairment of autophagic flux via modulation of the mTORC1/TFEB pathway. Graphic Abstract: A graphic abstract is available for this article.

Extracellular vesicles from human embryonic stem cell-derived cardiovascular progenitor cells promote cardiac infarct healing through reducing cardiomyocyte death and promoting angiogenesis.

Human pluripotent stem cells (hPSCs)-derived cardiovascular progenitor cells (CVPCs) are a promising source for myocardial repair, while the mechanisms remain largely unknown. Extracellular vesicles (EVs) are known to mediate cell-cell communication, however, the efficacy and mechanisms of hPSC-CVPC-secreted EVs (hCVPC-EVs) in the infarct healing when given at the acute phase of myocardial infarction (MI) are unknown. Here, we report the cardioprotective effects of the EVs secreted from hESC-CVPCs under normoxic (EV-N) and hypoxic (EV-H) conditions in the infarcted heart and the long noncoding RNA (lncRNA)-related mechanisms. The hCVPC-EVs were confirmed by electron microscopy, nanoparticle tracking, and immunoblotting analysis. Injection of hCVPC-EVs into acutely infracted murine myocardium significantly improved cardiac function and reduced fibrosis at day 28 post MI, accompanied with the improved vascularization and cardiomyocyte survival at border zones. Consistently, hCVPC-EVs enhanced the tube formation and migration of human umbilical vein endothelial cells (HUVECs), improved the cell viability, and attenuated the lactate dehydrogenase release of neonatal rat cardiomyocytes (NRCMs) with oxygen glucose deprivation (OGD) injury. Moreover, the improvement of the EV-H in cardiomyocyte survival and tube formation of HUVECs was significantly better than these in the EV-N. RNA-seq analysis revealed a high abundance of the lncRNA MALAT1 in the EV-H. Its abundance was upregulated in the infarcted myocardium and cardiomyocytes treated with hCVPC-EVs. Overexpression of human MALAT1 improved the cell viability of NRCM with OGD injury, while knockdown of MALAT1 inhibited the hCVPC-EV-promoted tube formation of HUVECs. Furthermore, luciferase activity assay, RNA pull-down, and manipulation of miR-497 levels showed that MALAT1 improved NRCMs survival and HUVEC tube formation through targeting miR-497. These results reveal that hCVPC-EVs promote the infarct healing through improvement of cardiomyocyte survival and angiogenesis. The cardioprotective effects of hCVPC-EVs can be enhanced by hypoxia-conditioning of hCVPCs and are partially contributed by MALAT1 via targeting the miRNA.

Cardioprotection of post-ischemic moderate ROS against ischemia/reperfusion via STAT3-induced the inhibition of MCU opening.

Enhanced reactive oxygen species (ROS) at the beginning of reperfusion activated signal transducer and activator of transcription 3 (STAT3) in intermittent hypobaric hypoxia (IHH)-afforded cardioprotection against ischemia/reperfusion (I/R). However, its mechanism remains largely unknown. This study aimed to investigate the role and the downstream of STAT3 in exogenous enhanced post-ischemic ROS-induced cardioprotection using the model of moderate hydrogen peroxide postconditioning (H2O2PoC) mimicking endogenous ROS in IHH. Moderate H2O2PoC not only improved the post-ischemic myocardial contractile recovery and reduced the infarct size in isolated rat I/R hearts, but also alleviated mitochondrial calcium overload and ameliorated Ca2+ transients, cell contraction, and mitochondrial membrane potential in rat I/R cardiomyocytes. However, the cardioprotective effects of moderate H2O2PoC were abrogated by Janus kinase 2 (JAK2)/STAT3 inhibitor AG490 in rat hearts as well as adenovirus-delivered short hairpin RNA specific for STAT3 and the opener of mitochondrial calcium uniporter (MCU) spermine in rat cardiomyocytes. Notably, the moderate H2O2PoC-afforded cardioprotection abrogated by spermine could be rescued by STAT3 over-expression with adenovirus in rat I/R cardiomyocytes. Besides, moderate H2O2PoC enhanced mitochondrial STAT3 expression during I/R. A co-localization/interaction of STAT3 or phospho-STAT3ser727 and MCU was observed in rat cardiomyocytes with moderate H2O2PoC at 5 and 30 min of reperfusion but not in rat I/R cardiomyocytes. Further, STAT3 interacted with the N-terminal domain (NTD) of MCU in rat cardiomyocytes with moderate H2O2PoC. These findings indicated that post-ischemic moderate ROS activate STAT3 against cardiac I/R by inhibiting MCU opening via its interaction with the NTD of MCU to alleviate mitochondrial calcium overload.

Berbamine postconditioning protects the heart from ischemia/reperfusion injury through modulation of autophagy.

Pretreatment of berbamine protects the heart from ischemia/reperfusion (I/R) injury. However it is unknown whether it has cardioprotection when given at the onset of reperfusion (postconditioning (PoC)), a protocol with more clinical impact. Autophagy is upregulated in I/R myocardium and exacerbates cardiomyocyte death during reperfusion. However, it is unknown whether the autophagy during reperfusion is regulated by berbamine. Here we investigated whether berbamine PoC (BMPoC) protects the heart through regulation of autophagy by analyzing the effects of BMPoC on infarct size and/or cell death, functional recovery and autophagy in perfused rat hearts and isolated cardiomyocytes subjected to I/R. Berbamine from 10 to 100 nM given during the first 5 min of reperfusion concentration-dependently improved post-ischemic myocardial function and attenuated cell death. Similar protections were observed in cardiomyocytes subjected to simulated I/R. Meanwhile, BMPoC prevented I/R-induced impairment of autophagosome processing in cardiomyocytes, characterized by increased LC3-II level and GFP-LC3 puncta, and decreased p62 degradation. Besides, lysosomal inhibitor chloroquine did not induce additional increase of LC3-II and P62 abundance after I/R but it reversed the effects of BMPoC in those parameters in cardiomyocytes, suggesting that I/R-impaired autophagic flux is restored by BMPoC. Moreover, I/R injury was accompanied by enhanced expression of Beclin 1, which was significantly inhibited by BMPoC. In vitro and in vivo adenovirus-mediated knockdown of Beclin 1 in myocardium and cardiomyocytes restored I/R-impaired autophagosome processing, associated with an improvement of post-ischemic recovery of myocardial contractile function and a reduction of cell death, but it did not have additive effects to BMPoC. Conversely, overexpression of Beclin 1 abolished the cardioprotection of BMPoC as did by overexpression of an essential autophagy gene Atg5. Furthermore, BMPoC-mediated cardioprotection was abolished by a specific Akt1/2 inhibitor A6730. Our results demonstrate that BMPoC confers cardioprotection by modulating autophagy during reperfusion through the activation of PI3K/Akt signaling pathway.

ROS generated during early reperfusion contribute to intermittent hypobaric hypoxia-afforded cardioprotection against postischemia-induced Ca(2+) overload and contractile dysfunction via the JAK2/STAT3 pathway.

Moderate enhanced reactive oxygen species (ROS) during early reperfusion trigger the cardioprotection against ischemia/reperfusion (I/R) injury, while the mechanism is largely unknown. Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) contributes to the cardioprotection but whether it is activated by ROS and how it regulates Ca(2+) homeostasis remain unclear. Here we investigated whether the ROS generated during early reperfusion protect the heart/cardiomyocyte against I/R-induced Ca(2+) overload and contractile dysfunction via the activation of JAK2/STAT3 signaling pathway by using a cardioprotective model of intermittent hypobaric hypoxia (IHH) preconditioning. IHH improved the postischemic recovery of myocardial contractile performance in isolated rat I/R hearts as well as Ca(2+) homeostasis and cell contraction in simulated I/R cardiomyocytes. Meanwhile, IHH enhanced I/R-increased STAT3 phosphorylation at tyrosine 705 in the nucleus and reversed I/R-suppressed STAT3 phosphorylation at serine 727 in the nucleus and mitochondria during reperfusion. Moreover, IHH improved I/R-suppressed sarcoplasmic reticulum (SR) Ca(2+)-ATPase 2 (SERCA2) activity, enhanced I/R-increased Bcl-2 expression, and promoted the co-localization and interaction of Bcl-2 with SERCA2 during reperfusion. These effects were abolished by scavenging ROS with N-(2-mercaptopropionyl)-glycine (2-MPG) and/or by inhibiting JAK2 with AG490 during the early reperfusion. Furthermore, IHH-improved postischemic SERCA2 activity and Ca(2+) homeostasis as well as cell contraction were reversed after Bcl-2 knockdown by short hairpin RNA. In addition, the reversal of the I/R-suppressed mitochondrial membrane potential by IHH was abolished by 2-MPG and AG490. These results indicate that during early reperfusion the ROS/JAK2/STAT3 pathways play a crucial role in (i) the IHH-maintained intracellular Ca(2+) homeostasis via the improvement of postischemic SERCA2 activity through the increase of SR Bcl-2 and its interaction with SERCA2; and (ii) the IHH-improved mitochondrial function.

Uncoupling protein 3 mediates H2O2 preconditioning-afforded cardioprotection through the inhibition of MPTP opening.

AIMS: Uncoupling protein 3 (UCP3), located in the mitochondrial inner membrane, is cardioprotective, but its mechanisms of preserving mitochondrial function during ischaemia/reperfusion (I/R) are not fully understood. This study investigated whether UCP3 mediates/mimics the cardioprotection of H2O2 preconditioning (H2O2PC) against I/R injury and the downstream pathway that mediates H2O2PC- and UCP3-afforded cardioprotection. METHODS AND RESULTS: H2O2PC at 20 µM for 5 min significantly improved post-ischaemic functional recovery and reduced lactate dehydrogenase (LDH) release and infarct size with concurrently up-regulated UCP3 expressions in perfused rat hearts subjected to global no-flow I/R. These protections were blocked by UCP3 knockdown with short hairpin RNA but mimicked by UCP3 overexpression. Consistently, H2O2PC-attenuated I/R-induced cytosolic and mitochondrial Ca(2+) overload, Ca(2+) transient suppression, mitochondrial reactive oxygen species burst, and loss of mitochondrial inner membrane potential were reversed by UCP3 knockdown but mimicked by UCP3 overexpression. Moreover, co-immunoprecipitation assay revealed an interaction of UCP3 with the mitochondrial permeability transition pore (mPTP) component, adenine nucleotide translocator (ANT), while the cardioprotection induced by H2O2PC- and UCP3 overexpression in mitochondria, cardiac function, and cell survival was abolished by atractyloside, a mPTP opener binding to ANT, and partially inhibited by a PI3K/Akt inhibitor wortmannin. Furthermore, H2O2PC up-regulated the phosphorylation of Akt, and glycogen synthase kinase 3ß was blocked by UCP3 knockdown but mimicked by UCP3 overexpression. CONCLUSION: UCP3 mediates the cardioprotection of H2O2PC against I/R injury by preserving the mitochondrial function through inhibiting mPTP opening via the interaction with ANT and the PI3K/Akt pathway. Our findings reveal novel mechanisms of UCP3 in the cardioprotection.

Degradation of cardiac myosin light chain kinase by matrix metalloproteinase-2 contributes to myocardial contractile dysfunction during ischemia/reperfusion.

Although ischemia/reperfusion (I/R)-induced myocardial contractile dysfunction is associated with a prominent decrease in myofilament Ca(2+) sensitivity, the underlying mechanisms have not yet been fully clarified. Phosphorylation of ventricular myosin light chain 2 (MLC-2v) facilitates actin-myosin interactions and enhances contractility, however, its level and regulation by cardiac MLC kinase (cMLCK) and cMLC phosphatase (cMLCP) in I/R hearts are debatable. In this study, the levels and/or effects of MLC-2v phosphorylation, cMLCK, cMLCP, and proteases during I/R were determined. Global myocardial I/R-suppressed cardiac performance in isolated rat hearts was concomitant with decreases of MLC-2v phosphorylation, myofibrillar Ca(2+)-stimulated ATPase activity, and cMLCK content, but not cMLCP proteins. Consistently, simulated I/R in isolated cardiomyocytes inhibited cell shortening, Ca(2+) transients, MLC-2v phosphorylation, and myofilament sensitivity to Ca(2+). These observations were reversed by cMLCK overexpression, while the specific cMLCK knockdown by short hairpin RNA (shRNA) had the opposite effect. Moreover, the inhibition of matrix metalloproteinase-2 (MMP-2, a zinc-dependent endopeptidase) reversed IR-decreased cMLCK, MLC-2v phosphorylation, myofibrillar Ca(2+)-stimulated ATPase activity, myocardial contractile function, and myofilament sensitivity to Ca(2+), while the inhibition or knockdown of cMLCK by ML-9 or specific shRNA abolished MMP-2 inhibition-induced cardioprotection. Finally, the co-localization in cardiomyocytes and interaction in vivo of MMP-2 and cMLCK were observed. Purified recombinant rat cMLCK was concentration- and time-dependently degraded by rat MMP-2 in vitro, and this was prevented by the inhibition of MMP-2. These findings reveal that the I/R-activated MMP-2 leads to the degradation of cMLCK, resulting in a reduction of MLC-2v phosphorylation, and myofibrillar Ca(2+)-stimulated ATPase activity, which subsequently suppresses myocardial contractile function through a decrease of myofilament Ca(2+) sensitivity.