Improved Cardiomyocyte Survival Research Articles

Sensory Neurons Release Cardioprotective Factors in an In Vitro Ischemia Model.

Sensory neurons densely innervate the myocardium. The role of their sensing and response to acute and prolonged ischemia is largely unclear. In a cellular model of ischemia-reperfusion injury, the presence of sensory neurons increases cardiomyocyte survival. Here, after the exclusion of classical neurotransmitter release, and measurement of cytokine release, we modified the experiment from a direct co-culture of primary murine cardiomyocytes and sensory neurons to a transfer of the supernatant. Sensory neurons were exposed to ischemia and the resulting conditioned supernatant was transferred onto cardiomyocytes. This approach largely increased the tolerance of cardiomyocytes to ischemia and reperfusion. Towards the identification of the mechanism, it was demonstrated that after ten-fold dilution, the conditioned solution lost its protective effect. The effect remained after removal of extracellular vesicles by ultracentrifugation, and was not affected by exposure to protease activity, and fractionation pointed towards a hydrophilic agent. Solutions conditioned by HEK293t cells or 3T3 fibroblasts also increase cardiomyocyte survival, but to a lower degree. A metabolomic search identified 64 at least two-fold changed metabolites and lipids. Many of these could be identified and are involved in essential cellular functions. In the presented model for ischemia-reperfusion, sensory neurons secrete one or more cardioprotective substances that can improve cardiomyocyte survival.

Laminin Alpha 2 Enhances the Protective Effect of Exosomes on Human iPSC-Derived Cardiomyocytes in an In Vitro Ischemia-Reoxygenation Model.

Ischemic heart disease, a leading cause of death worldwide, manifests clinically as myocardial infarction. Contemporary therapies using mesenchymal stromal cells (MSCs) and their derivative (exosomes, EXOs) were developed to decrease the progression of cell damage during ischemic injury. Laminin alpha 2 (LAMA2) is an important extracellular matrix protein of the heart. Here, we generated MSC-derived exosomes cultivated under LAMA2 coating to enhance human-induced pluripotent stem cell (hiPSC)-cardiomyocyte recognition of LAMA2-EXOs, thus, increasing cell protection during ischemia reoxygenation. We mapped the mRNA content of LAMA2 and gelatin-EXOs and identified 798 genes that were differentially expressed, including genes associated with cardiac muscle development and extracellular matrix organization. Cells were treated with LAMA2-EXOs 2 h before a 4 h ischemia period (1% O2, 5% CO2, glucose-free media). LAMA2-EXOs had a two-fold protective effect compared to non-treatment on plasma membrane integrity and the apoptosis activation pathway; after a 1.5 h recovery period (20% O2, 5% CO2, cardiomyocyte-enriched media), cardiomyocytes treated with LAMA2-EXOs showed faster recovery than did the control group. Although EXOs had a protective effect on endothelial cells, there was no LAMA2-enhanced protection on these cells. This is the first report of LAMA2-EXOs used to treat cardiomyocytes that underwent ischemia-reoxygenation injury. Overall, we showed that membrane-specific EXOs may help improve cardiomyocyte survival in treating ischemic cardiovascular disease.

Bioenergetic Metabolism Modulatory Peptide Hydrogel for Cardiac Protection and Repair After Myocardial Infarction

AbstractMyocardial infarction (MI) remains a major threat to human health due to the limited energy supply, disordered cell metabolism, massive cardiomyocyte death, and restricted regeneration. Although currently available therapies may relieve myocardial damage, restoring the dysregulated energy metabolism to normal levels has not yet been achieved. MOTS‐c has recently been identified as a regulator of biological metabolism to combat aging; however, its role in reprogramming cardiac metabolism remains to be elucidated. Here, MOTS‐c is chemically conjugated to self‐assembling Q11 peptide to fabricate an injectable hydrogel (MQgel) aimed to improve mitochondria function and cardiomyocyte metabolism post‐MI. It is observed that MQgel effectively protects mitochondria from oxidative damage and normalized cardiomyocyte metabolism, including glucose uptake, glycolysis, and the tricarboxylic acid (TCA) cycle, thereby inhibiting cardiomyocyte death and enhancing cardiomyocyte activity. In a rat MI model, intramyocardial injection of MQgel successfully minimizes the infarct area and fibrosis, promotes angiogenesis, suppresses myocardial hypertrophy, and improves cardiomyocyte survival and metabolic enzyme activity, all of which collaboratively attenuate the maladaptive cardiac remodeling and boost cardiac function and tissue repair. The findings suggest that the self‐assembled mitochondria metabolism‐regulatory peptide hydrogel effectively treats MI, and cellular bioenergy modulation provides a new therapeutic approach for tissue repair after injury.

Extracellular Matrix/Glycopeptide Hybrid Hydrogel as an Immunomodulatory Niche for Endogenous Cardiac Repair after Myocardial Infarction.

The treatment of myocardial infarction (MI) remains a substantial challenge due to excessive inflammation, massive cell death, and restricted regenerative potential, leading to maladaptive healing process and eventually heart failure. Current strategies of regulating inflammation or improving cardiac tissue regeneration have limited success. Herein, a hybrid hydrogel coassembled by acellular cardiac extracellular matrix (ECM) and immunomodulatory glycopeptide is developed for endogenous tissue regeneration after MI. The hydrogel constructs a niche recapitulating the architecture of native ECM for attracting host cell homing, controlling macrophage differentiation via glycopeptide unit, and promoting endotheliocyte proliferation by enhancing the macrophage-endotheliocyte crosstalk, which coordinate the innate healing mechanism for cardiac tissue regeneration. In a rodent MI model, the hybrid hydrogel successfully orchestrates a proreparative response indicated by enhanced M2 macrophage polarization, increased angiogenesis, and improved cardiomyocyte survival, which alleviates infarct size, improves wall thicknesses, and enhances cardiac contractility. Furthermore, the safety and effectiveness of the hydrogel are demonstrated in a porcine MI model, wherein proteomics verifies the regulation of immune response, proangiogenesis, and accelerated healing process. Collectively, the injectable composite hydrogel serving as an immunomodulatory niche for promoting cell homing and proliferation, inflammation modulation, tissue remodeling, and function restoration provides an effective strategy for endogenous cardiac repair.

Labdane conjugates protect cardiomyocytes from doxorubicin-induced cardiotoxicity.

The cardiovascular side effects associated with doxorubicin (DOX), a wide spectrum anticancer drug, have limited its clinical application. Therefore, to explore novel strategies with cardioprotective effects, a series of new labdane conjugates were prepared (6a-6cand8a-8d) from the natural diterpene labdanodiol (1). These hybrid compounds contain anti-inflammatory privileged structures such as naphthalimide, naphthoquinone, and furanonaphthoquinone. Biological activity of these conjugates against DOX-induced cardiotoxicity was tested in vitro and the potential molecular mechanisms of protective effects were explored in H9c2 cardiomyocytes. Three compounds 6c, 8a, and 8b significantly improved cardiomyocyte survival, via inhibition of reactive oxygen species-mediated mitogen-activated protein kinasesignaling pathways (extracellular signal-regulated kinase and c-Jun N-terminal kinase) and autophagy mediated by Akt activation. Some structure-activity relationships were outlined, and the best activity was achieved with the labdane-furonaphthoquinone conjugate 8a having anN-cyclohexyl substituent. The findings of this study pave the way for further investigations to obtain more compounds with potential cardioprotective activity.

Protective effect of HINT2 on mitochondrial function via repressing MCU complex activation attenuates cardiac microvascular ischemia\u2013reperfusion injury

Current evidence indicates that coronary microcirculation is a key target for protecting against cardiac ischemia–reperfusion (I/R) injury. Mitochondrial calcium uniporter (MCU) complex activation and mitochondrial calcium ([Ca2+]m) overload are underlying mechanisms involved in cardiovascular disease. Histidine triad nucleotide-binding 2 (HINT2) has been reported to modulate [Ca2+]m via the MCU complex, and our previous work demonstrated that HINT2 improved cardiomyocyte survival and preserved heart function in mice with cardiac ischemia. This study aimed to explore the benefits of HINT2 on cardiac microcirculation in I/R injury with a focus on mitochondria, the MCU complex, and [Ca2+]m overload in endothelial cells. The present work demonstrated that HINT2 overexpression significantly reduced the no-reflow area and improved microvascular perfusion in I/R-injured mouse hearts, potentially by promoting endothelial nitric oxide synthase (eNOS) expression and phosphorylation. Microvascular barrier function was compromised by reperfusion injury, but was repaired by HINT2 overexpression via inhibiting VE-Cadherin phosphorylation at Tyr731 and enhancing the VE-Cadherin/β-Catenin interaction. In addition, HINT2 overexpression inhibited the inflammatory response by suppressing vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Mitochondrial fission occurred in cardiac microvascular endothelial cells (CMECs) subjected to oxygen–glucose deprivation/reoxygenation (OGD/R) injury and resulted in mitochondrial dysfunction and mitochondrion-dependent apoptosis, the effects of which were largely relieved by HINT2 overexpression. Additional experiments confirmed that [Ca2+]m overload was an initiating factor for mitochondrial fission and that HINT2 suppressed [Ca2+]m overload via modulation of the MCU complex through directly interacting with MCU in CMECs. Regaining [Ca2+]m overload by spermine, an MCU agonist, abolished all the protective effects of HINT2 on OGD/R-injured CMECs and I/R-injured cardiac microcirculation. In conclusion, the present report demonstrated that HINT2 overexpression inhibited MCU complex-mitochondrial calcium overload-mitochondrial fission and apoptosis pathway, and thereby attenuated cardiac microvascular ischemia–reperfusion injury.

Cardiomyocyte Protection by Hibernating Brown Bear Serum: Toward the Identification of New Protective Molecules Against Myocardial Infarction.

Ischemic heart disease remains one of the leading causes of death worldwide. Despite intensive research on the treatment of acute myocardial infarction, no effective therapy has shown clinical success. Therefore, novel therapeutic strategies are required to protect the heart from reperfusion injury. Interestingly, despite physical inactivity during hibernation, brown bears (Ursus arctos) cope with cardiovascular physiological conditions that would be detrimental to humans. We hypothesized that bear serum might contain circulating factors that could provide protection against cell injury. In this study, we sought to determine whether addition of bear serum might improve cardiomyocyte survival following hypoxia–reoxygenation. Isolated mouse cardiomyocytes underwent 45 min of hypoxia followed by reoxygenation. At the onset of reoxygenation, cells received fetal bovine serum (FBS; positive control), summer (SBS) or winter bear serum (WBS), or adult serums of other species, as indicated. After 2 h of reoxygenation, propidium iodide staining was used to evaluate cell viability by flow cytometry. Whereas, 0.5% SBS tended to decrease reperfusion injury, 0.5% WBS significantly reduced cell death, averaging 74.04 ± 7.06% vs. 79.20 ± 6.53% in the FBS group. This cardioprotective effect was lost at 0.1%, became toxic above 5%, and was specific to the bear. Our results showed that bear serum exerts a therapeutic effect with an efficacy threshold, an optimal dose, and a toxic effect on cardiomyocyte viability after hypoxia–reoxygenation. Therefore, the bear serum may be a potential source for identifying new therapeutic molecules to fight against myocardial reperfusion injury and cell death in general.

CGMP and mitochondrial K+ channels-Compartmentalized but closely connected in cardioprotection.

The 3',5'-cGMP pathway triggers cytoprotective responses and improves cardiomyocyte survival during myocardial ischaemia and reperfusion (I/R) injury. These beneficial effects were attributed to NO-sensitive GC induced cGMP production leading to activation of cGMP-dependent protein kinase I (cGKI). cGKI in turn phosphorylates many substrates, which eventually facilitate opening of mitochondrial ATP-sensitive potassium channels (mitoKATP ) and Ca2+ -activated potassium channels of the BK type (mitoBK). Accordingly, agents activating mitoKATP or mitoBK provide protection against I/R-induced damages. Here, we provide an up-to-date summary of the infarct-limiting actions exhibited by the GC/cGMP axis and discuss how mitoKATP and mitoBK, which are present at the inner mitochondrial membrane, confer mito- and cytoprotective effects on cardiomyocytes exposed to I/R injury. In view of this, we believe that the functional connection between the cGMP cascade and mitoK+ channels should be exploited further as adjunct to reperfusion therapy in myocardial infarction. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

Cardiomyocytes stimulate angiogenesis after ischemic injury in a ZEB2-dependent manner

The disruption in blood supply due to myocardial infarction is a critical determinant for infarct size and subsequent deterioration in function. The identification of factors that enhance cardiac repair by the restoration of the vascular network is, therefore, of great significance. Here, we show that the transcription factor Zinc finger E-box-binding homeobox 2 (ZEB2) is increased in stressed cardiomyocytes and induces a cardioprotective cross-talk between cardiomyocytes and endothelial cells to enhance angiogenesis after ischemia. Single-cell sequencing indicates ZEB2 to be enriched in injured cardiomyocytes. Cardiomyocyte-specific deletion of ZEB2 results in impaired cardiac contractility and infarct healing post-myocardial infarction (post-MI), while cardiomyocyte-specific ZEB2 overexpression improves cardiomyocyte survival and cardiac function. We identified Thymosin β4 (TMSB4) and Prothymosin α (PTMA) as main paracrine factors released from cardiomyocytes to stimulate angiogenesis by enhancing endothelial cell migration, and whose regulation is validated in our in vivo models. Therapeutic delivery of ZEB2 to cardiomyocytes in the infarcted heart induces the expression of TMSB4 and PTMA, which enhances angiogenesis and prevents cardiac dysfunction. These findings reveal ZEB2 as a beneficial factor during ischemic injury, which may hold promise for the identification of new therapies.

Zinc-dependent changes in oxidative and endoplasmic reticulum stress during cardiomyocyte hypoxia/reoxygenation.

Myocardial zinc dyshomeostasis is associated with caspase-3 activation, ErbB2 degradation and apoptosis during hypoxia/reoxygenation. Zinc pyrithione replenishes intracellular zinc, suppresses caspase-3, augments ErbB2 levels and improves cell survival. We hypothesize that zinc is capable of modulating redox and endoplasmic reticulum (ER) stress in the setting of cardiomyocyte hypoxia-reoxygenation. Hypoxia/reoxygenation lowered intracellular zinc, increased ER as well as oxidative stress in H9c2 cells, both of which were effectively attenuated by zinc supplementation. Silencing of gp91phox attenuated oxidative and ER stress, decreased caspase-3 activation and improved cell survival. Mimicking the oxidative insult using 50μM H2O2 increased the caspase-3 activity that correlated with decreased ErbB2 levels, concomitant with augmented ER stress. N-acetyl cysteine (NAC) administration completely suppressed ER stress as well as caspase-3 activity. Zinc depletion using TPEN also resulted in lowered ErbB2 and increased apoptosis, along with NOX2 mRNA upregulation, increased oxidative and ER stress. Repletion with zinc suppressed NOX2 mRNA, lowered oxidative as well as ER stress and decreased cell death. These results suggest that zinc dyshomeostasis, along with oxidative stress contribute to the unfolded protein response during myocardial H/R and that zinc replenishment corrects zinc homeostasis, alleviates associated stress and improves cardiomyocyte survival.

Pharmacological inhibition of Hippo pathway, with the novel kinase inhibitor XMU-MP-1, protects the heart against adverse effects during pressure overload.

Background and PurposeThe Hippo pathway has emerged as a potential therapeutic target to control pathological cardiac remodelling. The core components of the Hippo pathway, mammalian Ste‐20 like kinase 1 (Mst1) and mammalian Ste‐20 like kinase 2 (Mst2), modulate cardiac hypertrophy, apoptosis, and fibrosis. Here, we study the effects of pharmacological inhibition of Mst1/2 using a novel inhibitor XMU‐MP‐1 in controlling the adverse effects of pressure overload‐induced hypertrophy.Experimental ApproachWe used cultured neonatal rat cardiomyocytes (NRCM) and C57Bl/6 mice with transverse aortic constriction (TAC) as in vitro and in vivo models, respectively, to test the effects of XMU‐MP‐1 treatment. We used luciferase reporter assays, western blots and immunofluorescence assays in vitro, with echocardiography, qRT‐PCR and immunohistochemical methods in vivo.Key ResultsXMU‐MP‐1 treatment significantly increased activity of the Hippo pathway effector yes‐associated protein and inhibited phenylephrine‐induced hypertrophy in NRCM. XMU‐MP‐1 improved cardiomyocyte survival and reduced apoptosis following oxidative stress. In vivo, mice 3 weeks after TAC, were treated with XMU‐MP‐1 (1 mg·kg−1) every alternate day for 10 further days. XMU‐MP‐1‐treated mice showed better cardiac contractility than vehicle‐treated mice. Cardiomyocyte cross‐sectional size and expression of the hypertrophic marker, brain natriuretic peptide, were reduced in XMU‐MP‐1‐treated mice. Improved heart function in XMU‐MP‐1‐treated mice with TAC, was accompanied by fewer TUNEL positive cardiomyocytes and lower levels of fibrosis, suggesting inhibition of cardiomyocyte apoptosis and decreased fibrosis.Conclusions and ImplicationsThe Hippo pathway inhibitor, XMU‐MP‐1, reduced cellular hypertrophy and improved survival in cultured cardiomyocytes and, in vivo, preserved cardiac function following pressure overload.

Inhibition of cardiomyocyte Sprouty1 protects from cardiac ischemia\u2013reperfusion injury

Sprouty1 (Spry1) is a negative modulator of receptor tyrosine kinase signaling, but its role in cardiomyocyte survival has not been elucidated. The aim of this study was to investigate the potential role of cardiomyocyte Spry1 in cardiac ischemia–reperfusion (I/R) injury. Infarct areas of mouse hearts showed an increase in Spry1 protein expression, which localized to cardiomyocytes. To investigate if cardiomyocyte Spry1 regulates I/R injury, 8-week-old inducible cardiomyocyte Spry1 knockout (Spry1 cKO) mice and control mice were subjected to cardiac I/R injury. Spry1 cKO mice showed reduction in release of cardiac troponin I and reduced infarct size after I/R injury compared to control mice. Similar to Spry1 knockdown in cardiomyocytes in vivo, RNAi-mediated Spry1 silencing in isolated cardiomyocytes improved cardiomyocyte survival following simulated ischemia injury. Mechanistically, Spry1 knockdown induced cardiomyocyte extracellular signal-regulated kinase (ERK) phosphorylation in healthy hearts and isolated cardiomyocytes, and enhanced ERK phosphorylation after I/R injury. Spry1-deficient cardiomyocytes showed better preserved mitochondrial membrane potential following ischemic injury and an increase in levels of phosphorylated ERK and phosphorylated glycogen synthase kinase-3β (GSK-3β) in mitochondria of hypoxic cardiomyocytes. Overexpression of constitutively active GSK-3β abrogated the protective effect of Spry1 knockdown. Moreover, pharmacological inhibition of GSK-3β protected wild-type cardiomyocytes from cell death, but did not further protect Spry1-silenced cardiomyocytes from hypoxia-induced injury. Cardiomyocyte Spry1 knockdown promotes ERK phosphorylation and offers protection from I/R injury. Our findings indicate that Spry1 is an important regulator of cardiomyocyte viability during ischemia–reperfusion injury.

In Situ Gelling Scaffolds Loaded with Platelet Growth Factors to Improve Cardiomyocyte Survival after Ischemia.

Myocardial infarction is caused by prolonged ischemia and it is one of the main cause that leads to heart failures. The aim of the present work was the development of in situ gelling systems, based on poloxamer 407 (P407) or sodium alginate (Alg), loaded with platelet lysate (PL) to enhance cardiomyocyte survival after ischemia. Chondroitin sulfate (CS), a negatively charged glycosaminoglycan able to interact with different positively charged bioactive molecules, such as growth factors, was also investigated with both the systems. The gelation properties of both systems (viscosity, viscoelasticity, consistency by means of penetrometry, and injectability) were characterized in a physiological environment. In vitro evaluation of biocompatibility using fetal cardiac cells (cardiomyocytes and cardiac fibroblasts) demonstrated that the PL loaded alginate/chondroitin sulfate system retained the highest number of viable cells with equal distribution of the populations of cardiomyocytes and fibroblasts. Furthermore, the ability of the systems to improve cardiomyocyte survival after ischemia was also assessed. PL allowed for the highest degree of survival of cardiomyocytes after oxidative damage (simulating ischemic conditions due to MI) and both the Alg + CS PL and, to a greater extent, the PL alone demonstrated a considerable increase in survival of cardiomyocytes. In conclusion, an in situ gelling alginate-chondroitin sulfate system, loaded with platelet lysate, was able to improve the survival of cardiomyocytes after oxidative damage resulting in a promising system to improve cardiac cell viability after ischemia.

Role of TLR4/MyD88/NF-κB signaling pathway in coronary microembolization-induced myocardial injury prevented and treated with nicorandil

Coronary microembolization (CME) is a common complication during the treatment of acute coronary syndrome (ACS) and percutaneous coronary intervention (PCI). Nicorandil can be used to prevent myocardial injury after PCI to reduce the incidence of coronary no-reflow and slow flow, and play a role in myocardial protection, suggesting that its mechanism may be related to the inhibition of CME-induced inflammation of cardiomyocytes. However, the specific mechanism remains unclear. This study investigated the myocardial protective effects of nicorandil pretreatment on CME-induced myocardial injury and the specific mechanism of its inhibition of myocardial inflammation. An CME rat model exhibited CME-induced myocardial inflammation and the elevation of serum tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1β based on echocardiography, myocardial enzyme detection, hematoxylin and eosin (HE) and hematoxylin-basic fuchsin-picric acid (HBFP) stainings, ELISA, quantitative real-time PCR, and western blotting. Nicorandil treatment seven days before CME induction effectively inhibited myocardial inflammation, ameliorated myocardial injury, and improved cardiac function, mainly by inhibiting Toll-like receptor 4 (TLR4)-mediated myeloid differentiation primary response protein 88 (MyD88)-dependent nuclear factor-kappa B (NF-κB) signaling. Rat neonatal cardiomyocyte experiments further confirmed that nicorandil ameliorated lipopolysaccharide (LPS)-induced myocardial inflammation and improved cardiomyocyte survival. The specific mechanisms mainly involved the inhibition of TLR4/MyD88/NF-κB signaling and the reduction of the inflammatory cytokines TNF-α and IL-1β released from cardiomyocytes. In summary, nicorandil significantly protected cardiomyocytes from CME-induced myocardial injury mainly by inhibiting TLR4/MyD88/NF-κB signaling, thereby reducing the onset of CME-induced myocardial inflammation. This could be one of the important mechanisms for reducing postoperative myocardial injury via PCI-preoperative prophylactic treatment with nicorandil.

Large Cardiac Muscle Patches Engineered From Human Induced-Pluripotent Stem Cell-Derived Cardiac Cells Improve Recovery From Myocardial Infarction in Swine.

Here, we generated human cardiac muscle patches (hCMPs) of clinically relevant dimensions (4 cm × 2 cm × 1.25 mm) by suspending cardiomyocytes, smooth muscle cells, and endothelial cells that had been differentiated from human induced-pluripotent stem cells in a fibrin scaffold and then culturing the construct on a dynamic (rocking) platform. In vitro assessments of hCMPs suggest maturation in response to dynamic culture stimulation. In vivo assessments were conducted in a porcine model of myocardial infarction (MI). Animal groups included: MI hearts treated with 2 hCMPs (MI+hCMP, n=13), MI hearts treated with 2 cell-free open fibrin patches (n=14), or MI hearts with neither experimental patch (n=15); a fourth group of animals underwent sham surgery (Sham, n=8). Cardiac function and infarct size were evaluated by MRI, arrhythmia incidence by implanted loop recorders, and the engraftment rate by calculation of quantitative polymerase chain reaction measurements of expression of the human Y chromosome. Additional studies examined the myocardial protein expression profile changes and potential mechanisms of action that related to exosomes from the cell patch. The hCMPs began to beat synchronously within 1 day of fabrication, and after 7 days of dynamic culture stimulation, in vitro assessments indicated the mechanisms related to the improvements in electronic mechanical coupling, calcium-handling, and force generation, suggesting a maturation process during the dynamic culture. The engraftment rate was 10.9±1.8% at 4 weeks after the transplantation. The hCMP transplantation was associated with significant improvements in left ventricular function, infarct size, myocardial wall stress, myocardial hypertrophy, and reduced apoptosis in the periscar boarder zone myocardium. hCMP transplantation also reversed some MI-associated changes in sarcomeric regulatory protein phosphorylation. The exosomes released from the hCMP appeared to have cytoprotective properties that improved cardiomyocyte survival. We have fabricated a clinically relevant size of hCMP with trilineage cardiac cells derived from human induced-pluripotent stem cells. The hCMP matures in vitro during 7 days of dynamic culture. Transplantation of this type of hCMP results in significantly reduced infarct size and improvements in cardiac function that are associated with reduction in left ventricular wall stress. The hCMP treatment is not associated with significant changes in arrhythmogenicity.

HSPA12B Attenuated Acute Myocardial Ischemia/reperfusion Injury via Maintaining Endothelial Integrity in a PI3K/Akt/mTOR-dependent Mechanism.

Endothelial damage is a critical mediator of myocardial ischemia/reperfusion (I/R) injury. HSPA12B is an endothelial-cell-specifically expressed heat shock protein. However, the roles of HSPA12B in acute myocardial I/R injury is unknown. Here we reported that myocardial I/R upregulated HSPA12B expression in ventricular tissues, and endothelial overexpression of HSPA12B in transgenic mice (Tg) limited infarct size, attenuated cardiac dysfunction and improved cardiomyocyte survival compared with their wild type littermates. These improvements were accompanied with the diminished myocardial no-reflow phenomenon, decreased microvascular leakage, and better maintained endothelial tight junctions. The I/R-evoked neutrophil infiltration was also suppressed in Tg hearts compared with its wild type (WT) littermates. Moreover, Tg hearts exhibited the enhanced activation of PI3K/Akt//mTOR signaling following I/R challenge. However, pharmacological inhibition of PI3K abolished the HSPA12B-induced cardioprotection against myocardial I/R injury. The data demonstrate for the first time that the endothelial HSPA12B protected hearts against myocardial I/R injury. This cardioprotective action of HSPA12B was mediated, at least in part, by improving endothelial integrity in a PI3K/Akt/mTOR-dependent mechanism. Our study suggests that targeting endothelial HSPA12B could be an alternative approach for the management of patients with myocardial I/R injury.

Temporary, Systemic Inhibition of the WNT/β-Catenin Pathway promotes Regenerative Cardiac Repair following Myocardial Infarct.

The WNT/β-catenin pathway is temporarily activated in the heart following myocardial infarction (MI). Despite data from genetic models indicating both positive and negative roles for the WNT pathway depending on the model used, the effect of therapeutic inhibition of WNT pathway on post-injury outcome and the cellular mediators involved are not completely understood. Using a newly available, small molecule, GNF-6231, which averts WNT pathway activation by blocking secretion of all WNT ligands, we sought to investigate whether therapeutic inhibition of the WNT pathway temporarily after infarct can mitigate post injury cardiac dysfunction and fibrosis and the cellular mechanisms responsible for the effects. Pharmacologic inhibition of the WNT pathway by post-MI intravenous injection of GNF-6231 in C57Bl/6 mice significantly reduced the decline in cardiac function (Fractional Shortening at day 30: 38.71 ± 4.13% in GNF-6231 treated vs. 34.89 ± 4.86% in vehicle-treated), prevented adverse cardiac remodeling, and reduced infarct size (9.07 ± 3.98% vs. 17.18 ± 4.97%). WNT inhibition augmented proliferation of interstitial cells, particularly in the distal myocardium, inhibited apoptosis of cardiomyocytes, and reduced myofibroblast proliferation in the peri-infarct region. In vitro studies showed that WNT inhibition increased proliferation of Sca1+ cardiac progenitors, improved survival of cardiomyocytes, and inhibited collagen I synthesis by cardiac myofibroblasts. Systemic, temporary pharmacologic inhibition of the WNT pathway using an orally bioavailable drug immediately following MI resulted in improved function, reduced adverse remodeling and reduced infarct size in mice. Therapeutic WNT inhibition affected multiple aspects of infarct repair: it promoted proliferation of cardiac progenitors and other interstitial cells, inhibited myofibroblast proliferation, improved cardiomyocyte survival, and reduced collagen I gene expression by myofibroblasts. Our data point to a promising role for WNT inhibitory therapeutics as a new class of drugs to drive post-MI repair and prevent heart failure.

Micromanaging cardiac regeneration: Targeted delivery of microRNAs for cardiac repair and regeneration.

The loss of cardiomyocytes during injury and disease can result in heart failure and sudden death, while the adult heart has a limited capacity for endogenous regeneration and repair. Current stem cell-based regenerative medicine approaches modestly improve cardiomyocyte survival, but offer neglectable cardiomyogenesis. This has prompted the need for methodological developments that crease de novo cardiomyocytes. Current insights in cardiac development on the processes and regulatory mechanisms in embryonic cardiomyocyte differentiation provide a basis to therapeutically induce these pathways to generate new cardiomyocytes. Here, we discuss the current knowledge on embryonic cardiomyocyte differentiation and the implementation of this knowledge in state-of-the-art protocols to the direct reprogramming of cardiac fibroblasts into de novo cardiomyocytes in vitro and in vivo with an emphasis on microRNA-mediated reprogramming. Additionally, we discuss current advances on state-of-the-art targeted drug delivery systems that can be employed to deliver these microRNAs to the damaged cardiac tissue. Together, the advances in our understanding of cardiac development, recent advances in microRNA-based therapeutics, and innovative drug delivery systems, highlight exciting opportunities for effective therapies for myocardial infarction and heart failure.

TUG Mediates GLUT4 Translocation by AMP‐Activated Protein Kinase in the Heart

IntroductionIschemic heart disease, is mainly caused by reduced nutrient availability. Increasing nutrient uptake becomes a key approach to improve cardiomyocyte survival during the ischemia and reperfusion (I/R) period. TUG (tether containing a UBX domain, for GLUT4, 60 KDa) is a regulator of GLUT4 trafficking, which is cleaved to mobilize GLUT4 from intracellular compartments to the cell surface. The energy sensor AMP‐activated protein kinase (AMPK) is known to play an important cardioprotective role during myocardial I/R by regulating GLUT4 translocation. Antithrombin (AT) exhibits a cardioprotective function severed as AMPK activator. 
HypothesisTUG is one of the downstream targets of AMPK, which can be phosphorylated by hypoxia/ischemia‐induced as well as AT‐induced AMPK activation. 
MethodsIn vitro hypoxia chamber and in vivo I/R models were used to analyze AMPK/TUG interactions. Ex vivo isolated mouse heart perfusion Langendorff system was used to measure glucose metabolism. 
ResultsIn HL‐1 cardiomyocytes, TUG proteins were phosphorylated by activated AMPK during hypoxia. Moreover, TUG siRNA knockdown the TUG of HL‐1 cells caused significantly increased cell surface GLUT4 and glucose uptake. The ex vivo heart perfusion data demonstrated that AT triggered AMPK activation and significantly increase glucose uptake and GLUT4 translocation during I/R. Moreover, AT treatment increased abundance of a TUG cleavage product (42KDa) in response to I/R. All of these glucose transporter events are blunted in the AMPK kinase dead transgenic hearts. 
ConclusionsCardiac AMPK activation via AT during the I/R period stimulates TUG cleavage and causes the dissociation between TUG and GLUT4, followed by the enhanced glucose uptake.

CXCR4 attenuates cardiomyocytes mitochondrial dysfunction to resist ischaemia-reperfusion injury.

The chemokine (C-X-C motif) receptor 4 (CXCR4) is expressed on native cardiomyocytes and can modulate isolated cardiomyocyte contractility. This study examines the role of CXCR4 in cardiomyocyte response to ischaemia-reperfusion (I/R) injury. Isolated adult rat ventricular cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) to simulate I/R injury. In response to H/R injury, the decrease in CXCR4 expression was associated with dysfunctional energy metabolism indicated by an increased adenosine diphosphate/adenosine triphosphate (ADP/ATP) ratio. CXCR4-overexpressing cardiomyocytes were used to determine whether such overexpression (OE) can prevent bio-energetic disruption-associated cell death. CXCR4 OE was performed with adenoviral infection with CXCR4 encoding-gene or non-translated nucleotide sequence (Control). The increased CXCR4 expression was observed in cardiomyocytes post CXCR4-adenovirus transduction and this OE significantly reduced the cardiomyocyte contractility under basal conditions. Although the same extent of H/R-provoked cytosolic calcium overload was measured, the hydrogen peroxide-induced decay of mitochondrial membrane potential was suppressed in CXCR4 OE group compared with control group, and the mitochondrial swelling was significantly attenuated in CXCR4 group, implicating that CXCR4 OE prevents permeability transition pore opening exposure to overload calcium. Interestingly, this CXCR4-induced mitochondrial protective effect is associated with the enhanced signal transducer and activator of transcription 3 (expression in mitochondria. Consequently, in the presence of H/R, mitochondrial dysfunction was mitigated and cardiomyocyte death was decreased to 65% in the CXCR4 OE group as compared with the control group. I/R injury leads to the reduction in CXCR4 in cardiomyocytes associated with the dysfunctional energy metabolism, and CXCR4 OE can alleviate mitochondrial dysfunction to improve cardiomyocyte survival.