Podoplanin Positive Cell-derived Extracellular Vesicles Contribute to Cardiac Amyloidosis After Myocardial Infarction.

Background: Amyloidosis is a major long-term complication of chronic disease; however, whether it represents one of the complications of post-myocardial infarction (MI) is yet to be fully understood. Methods: Using wild-type and knocked-out MI mouse models and characterizing in vitro the exosomal communication between bone marrow-derived macrophages and activated mesenchymal stromal cells (MSC) isolated after MI, we investigated the mechanism behind Serum Amyloid A 3 (SAA3) protein overproduction in injured hearts. Results: Here, we show that amyloidosis occurs after MI and that amyloid fibers are composed of macrophage-derived SAA3 monomers. SAA3 overproduction in macrophages is triggered by exosomal communication from a subset of activated MSC, which, in response to MI, acquire the expression of a platelet aggregation-inducing type I transmembrane glycoprotein named Podoplanin (PDPN). Cardiac MSC PDPN+ communicate with and activate macrophages through their extracellular vesicles or exosomes. Specifically, MSC PDPN+ derived exosomes (MSC PDPN+ Exosomes) are enriched in SAA3 and exosomal SAA3 protein engages with Toll-like receptor 2 (TRL2) on macrophages, triggering an overproduction and impaired clearance of SAA3 proteins, resulting in aggregation of SAA3 monomers as rigid amyloid deposits in the extracellular space. The onset of amyloid fibers deposition alongside extra-cellular-matrix (ECM) proteins in the ischemic heart exacerbates the rigidity and stiffness of the scar, hindering the contractility of viable myocardium and overall impairing organ function. Using SAA3 and TLR2 deficient mouse models, we show that SAA3 delivered by MSC PDPN+ exosomes promotes post-MI amyloidosis. Inhibition of SAA3 aggregation via administration of a retro-inverso D-peptide, specifically designed to bind SAA3 monomers, prevents the deposition of SAA3 amyloid fibrils, positively modulates the scar formation, and improves heart function post-MI. Conclusion: Overall, our findings provide mechanistic insights into post-MI amyloidosis and suggest that SAA3 may be an attractive target for effective scar reversal after ischemic injury and a potential target in multiple diseases characterized by a similar pattern of inflammation and amyloid deposition. NOVELTY AND SIGNIFICANCE: What is known? Accumulation of rigid amyloid structures in the left ventricular wall impairs ventricle contractility.After myocardial infarction cardiac Mesenchymal Stromal Cells (MSC) acquire Podoplanin (PDPN) to better interact with immune cells.Amyloid structures can accumulate in the heart after chronic inflammatory conditions. What information does this article contribute? Whether accumulation of cumbersome amyloid structures in the ischemic scar impairs left ventricle contractility, and scar reversal after myocardial infarction (MI) has never been investigated.The pathophysiological relevance of PDPN acquirement by MSC and the functional role of their secreted exosomes in the context of post-MI cardiac remodeling has not been investigated.Amyloid structures are present in the scar after ischemia and are composed of macrophage-derived Serum Amyloid A (SAA) 3 monomers, although mechanisms of SAA3 overproduction is not established. SUMMARY OF NOVELTY AND SIGNIFICANCE: Here, we report that amyloidosis, a secondary phenomenon of an already preexisting and prolonged chronic inflammatory condition, occurs after MI and that amyloid structures are composed of macrophage-derived SAA3 monomers. Frequently studied cardiac amyloidosis are caused by aggregation of immunoglobulin light chains, transthyretin, fibrinogen, and apolipoprotein in a healthy heart as a consequence of systemic chronic inflammation leading to congestive heart failure with various types of arrhythmias and tissue stiffness. Although chronic MI is considered a systemic inflammatory condition, studies regarding the possible accumulation of amyloidogenic proteins after MI and the mechanisms involved in that process are yet to be reported. Here, we show that SAA3 overproduction in macrophages is triggered in a Toll-like Receptor 2 (TLR2)-p38MAP Kinase-dependent manner by exosomal communication from a subset of activated MSC, which, in response to MI, express a platelet aggregation-inducing type I transmembrane glycoprotein named Podoplanin. We provide the full mechanism of this phenomenon in murine models and confirm SAA3 amyloidosis in failing human heart samples. Moreover, we developed a retro-inverso D-peptide therapeutic approach, "DRI-R5S," specifically designed to bind SAA3 monomers and prevent post-MI aggregation and deposition of SAA3 amyloid fibrils without interfering with the innate immune response.

Tipifarnib Reduces Extracellular Vesicles and Protects From Heart Failure.

BACKGROUND: Heart failure (HF) is one of the leading causes of mortality worldwide. Extracellular vesicles, including small extracellular vesicles or exosomes, and their molecular cargo are known to modulate cell-to-cell communication during multiple cardiac diseases. However, the role of systemic extracellular vesicle biogenesis inhibition in HF models is not well documented and remains unclear. METHODS: We investigated the role of circulating exosomes during cardiac dysfunction and remodeling in a mouse transverse aortic constriction (TAC) model of HF. Importantly, we investigate the efficacy of tipifarnib, a recently identified exosome biogenesis inhibitor that targets the critical proteins (Rab27a [Ras associated binding protein 27a], nSMase2 [neutral sphingomyelinase 2], and Alix [ALG-2-interacting protein X]) involved in exosome biogenesis for this mouse model of HF. In this study, 10-week-old male mice underwent TAC surgery were randomly assigned to groups with and without tipifarnib treatment (10 mg/kg 3 times/wk) and monitored for 8 weeks, and a comprehensive assessment was conducted through performed echocardiographic, histological, and biochemical studies. RESULTS: TAC significantly elevated circulating plasma exosomes and markedly increased cardiac left ventricular dysfunction, cardiac hypertrophy, and fibrosis. Furthermore, injection of plasma exosomes from TAC mice induced left ventricular dysfunction and cardiomyocyte hypertrophy in uninjured mice without TAC. On the contrary, treatment of tipifarnib in TAC mice reduced circulating exosomes to baseline and remarkably improved left ventricular functions, hypertrophy, and fibrosis. Tipifarnib treatment also drastically altered the miRNA profile of circulating post-TAC exosomes, including miR 331-5p, which was highly downregulated both in TAC circulating exosomes and in TAC cardiac tissue. Mechanistically, miR 331-5p is crucial for inhibiting the fibroblast-to-myofibroblast transition by targeting HOXC8, a critical regulator of fibrosis. Tipifarnib treatment in TAC mice upregulated the expression of miR 331-5p that acts as a potent repressor for one of the fibrotic mechanisms mediated by HOXC8. CONCLUSIONS: Our study underscores the pathological role of exosomes in HF and fibrosis in response to pressure overload. Tipifarnib-mediated inhibition of exosome biogenesis and cargo sorting may serve as a viable strategy to prevent progressive cardiac remodeling in HF.

Epigenetic mechanisms regulate sex differences in cardiac reparative functions of bone marrow progenitor cells.

Historically, a lower incidence of cardiovascular diseases (CVD) and related deaths in women as compared with men of the same age has been attributed to female sex hormones, particularly estrogen and its receptors. Autologous bone marrow stem cell (BMSC) clinical trials for cardiac cell therapy overwhelmingly included male patients. However, meta-analysis data from these trials suggest a better functional outcome in postmenopausal women as compared with aged-matched men. Mechanisms governing sex-specific cardiac reparative activity in BMSCs, with and without the influence of sex hormones, remain unexplored. To discover these mechanisms, Male (M), female (F), and ovariectomized female (OVX) mice-derived EPCs were subjected to a series of molecular and epigenetic analyses followed by in vivo functional assessments of cardiac repair. F-EPCs and OVX EPCs show a lower inflammatory profile and promote enhanced cardiac reparative activity after intra-cardiac injections in a male mouse model of myocardial infarction (MI). Epigenetic sequencing revealed a marked difference in the occupancy of the gene repressive H3K9me3 mark, particularly at transcription start sites of key angiogenic and proinflammatory genes in M-EPCs compared with F-EPCs and OVX-EPCs. Our study unveiled that functional sex differences in EPCs are, in part, mediated by differential epigenetic regulation of the proinflammatory and anti-angiogenic gene CCL3, orchestrated by the control of H3K9me3 by histone methyltransferase, G9a/Ehmt2. Our research highlights the importance of considering the sex of donor cells for progenitor-based tissue repair.

GSK-3α aggravates inflammation, metabolic derangement, and cardiac injury post-ischemia/reperfusion.

Reperfusion after acute myocardial infarction further exaggerates cardiac injury and adverse remodeling. Irrespective of cardiac cell types, loss of specifically the α isoform of the protein kinase GSK-3 is protective in chronic cardiac diseases. However, the role of GSK-3α in clinically relevant ischemia/reperfusion (I/R)-induced cardiac injury is unknown. Here, we challenged cardiomyocyte-specific conditional GSK-3α knockout (cKO) and littermate control mice with I/R injury and investigated the underlying molecular mechanism using an in vitro GSK-3α gain-of-function model in AC16 cardiomyocytes post-hypoxia/reoxygenation (H/R). Analysis revealed a significantly lower percentage of infarct area in the cKO vs. control hearts post-I/R. Consistent with in vivo findings, GSK-3α overexpression promoted AC16 cardiomyocyte death post-H/R which was accompanied by an induction of reactive oxygen species (ROS) generation. Consistently, GSK-3α gain-of-function caused mitochondrial dysfunction by significantly suppressing mitochondrial membrane potential. Transcriptomic analysis of GSK-3α overexpressing cardiomyocytes challenged with hypoxia or H/R revealed that NOD-like receptor (NLR), TNF, NF-κB, IL-17, and mitogen-activated protein kinase (MAPK) signaling pathways were among the most upregulated pathways. Glutathione and fatty acid metabolism were among the top downregulated pathways post-H/R. Together, these observations suggest that loss of cardiomyocyte-GSK-3α attenuates cardiac injury post-I/R potentially through limiting the myocardial inflammation, mitochondrial dysfunction, and metabolic derangement. Therefore, selective inhibition of GSK-3α may provide beneficial effects in I/R-induced cardiac injury and remodeling. KEY MESSAGES: GSK-3α promotes cardiac injury post-ischemia/reperfusion (I/R). GSK-3α regulates inflammatory and metabolic pathways post-hypoxia/reoxygenation (H/R). GSK-3α overexpression upregulates NOD-like receptor (NLR), TNF, NF-kB, IL-17, and MAPK signaling pathways in cardiomyocytes post-H/R. GSK-3α downregulates glutathione and fatty acid metabolic pathways in cardiomyocytes post-H/R.

Nitrative Modification of Caveolin-3: A Novel Mechanism of Cardiac Insulin Resistance and a Potential Therapeutic Target Against Ischemic Heart Failure in Prediabetic Animals.

BACKGROUND: Myocardial insulin resistance is a hallmark of diabetic cardiac injury. However, the underlying molecular mechanisms remain unclear. Recent studies demonstrate that the diabetic heart is resistant to other cardioprotective interventions, including adiponectin and preconditioning. The "universal" resistance to multiple therapeutic interventions suggests impairment of the requisite molecule(s) involved in broad prosurvival signaling cascades. Cav (Caveolin) is a scaffolding protein coordinating transmembrane signaling transduction. However, the role of Cav3 in diabetic impairment of cardiac protective signaling and diabetic ischemic heart failure is unknown. METHODS: Wild-type and gene-manipulated mice were fed a normal diet or high-fat diet for 2 to 12 weeks and subjected to myocardial ischemia and reperfusion. Insulin cardioprotection was determined. RESULTS: Compared with the normal diet group, the cardioprotective effect of insulin was significantly blunted as early as 4 weeks of high-fat diet feeding (prediabetes), a time point where expression levels of insulin-signaling molecules remained unchanged. However, Cav3/insulin receptor-ß complex formation was significantly reduced. Among multiple posttranslational modifications altering protein/protein interaction, Cav3 (not insulin receptor-ß) tyrosine nitration is prominent in the prediabetic heart. Treatment of cardiomyocytes with 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride reduced the signalsome complex and blocked insulin transmembrane signaling. Mass spectrometry identified Tyr73 as the Cav3 nitration site. Phenylalanine substitution of Tyr73 (Cav3Y73F) abolished 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride-induced Cav3 nitration, restored Cav3/insulin receptor-ß complex, and rescued insulin transmembrane signaling. It is most important that adeno-associated virus 9-mediated cardiomyocyte-specific Cav3Y73F reexpression blocked high-fat diet-induced Cav3 nitration, preserved Cav3 signalsome integrity, restored transmembrane signaling, and rescued insulin-protective action against ischemic heart failure. Last, diabetic nitrative modification of Cav3 at Tyr73 also reduced Cav3/AdipoR1 complex formation and blocked adiponectin cardioprotective signaling. CONCLUSIONS: Nitration of Cav3 at Tyr73 and resultant signal complex dissociation results in cardiac insulin/adiponectin resistance in the prediabetic heart, contributing to ischemic heart failure progression. Early interventions preserving Cav3-centered signalsome integrity is an effective novel strategy against diabetic exacerbation of ischemic heart failure.

ß3AR-Dependent Brain-Derived Neurotrophic Factor (BDNF) Generation Limits Chronic Postischemic Heart Failure.

BACKGROUND: Loss of brain-derived neurotrophic factor (BDNF)/TrkB (tropomyosin kinase receptor B) signaling accounts for brain and cardiac disorders. In neurons, ß-adrenergic receptor stimulation enhances local BDNF expression. It is unclear if this occurs in a pathophysiological relevant manner in the heart, especially in the ß-adrenergic receptor-desensitized postischemic myocardium. Nor is it fully understood whether and how TrkB agonists counter chronic postischemic left ventricle (LV) decompensation, a significant unmet clinical milestone. METHODS: We conducted in vitro studies using neonatal rat and adult murine cardiomyocytes, SH-SY5Y neuronal cells, and umbilical vein endothelial cells. We assessed myocardial ischemia (MI) impact in wild type, ß3AR knockout, or myocyte-selective BDNF knockout (myoBDNF KO) mice in vivo (via coronary ligation [MI]) or in isolated hearts with global ischemia-reperfusion (I/R). RESULTS: In wild type hearts, BDNF levels rose early after MI (<24 hours), plummeting at 4 weeks when LV dysfunction, adrenergic denervation, and impaired angiogenesis ensued. The TrkB agonist, LM22A-4, countered all these adverse effects. Compared with wild type, isolated myoBDNF KO hearts displayed worse infarct size/LV dysfunction after I/R injury and modest benefits from LM22A-4. In vitro, LM22A-4 promoted neurite outgrowth and neovascularization, boosting myocyte function, effects reproduced by 7,8-dihydroxyflavone, a chemically unrelated TrkB agonist. Superfusing myocytes with the ß3AR-agonist, BRL-37344, increased myocyte BDNF content, while ß3AR signaling underscored BDNF generation/protection in post-MI hearts. Accordingly, the ß1AR blocker, metoprolol, via upregulated ß3ARs, improved chronic post-MI LV dysfunction, enriching the myocardium with BDNF. Last, BRL-37344-imparted benefits were nearly abolished in isolated I/R injured myoBDNF KO hearts. CONCLUSIONS: BDNF loss underscores chronic postischemic heart failure. TrkB agonists can improve ischemic LV dysfunction via replenished myocardial BDNF content. Direct cardiac ß3AR stimulation, or ß-blockers (via upregulated ß3AR), is another BDNF-based means to fend off chronic postischemic heart failure.

Beta-3 adrenergic receptor overexpression reverses aortic stenosis-induced heart failure and restores balanced mitochondrial dynamics.

Aortic stenosis (AS) is associated with left ventricular (LV) hypertrophy and heart failure (HF). There is a lack of therapies able to prevent/revert AS-induced HF. Beta3 adrenergic receptor (ß3AR) signaling is beneficial in several forms of HF. Here, we studied the potential beneficial effect of ß3AR overexpression on AS-induced HF. Selective ß3AR stimulation had a positive inotropic effect. Transgenic mice constitutively overexpressing human ß3AR in the heart (c-hß3tg) were protected from the development of HF in response to induced AS, and against cardiomyocyte mitochondrial dysfunction (fragmented mitochondria with remodeled cristae and metabolic reprogramming featuring altered substrate use). Similar beneficial effects were observed in wild-type mice inoculated with adeno-associated virus (AAV9) inducing cardiac-specific overexpression of human ß3AR before AS induction. Moreover, AAV9-hß3AR injection into wild-type mice at late disease stages, when cardiac hypertrophy and metabolic reprogramming are already advanced, reversed the HF phenotype and restored balanced mitochondrial dynamics, demonstrating the potential of gene-therapy-mediated ß3AR overexpression in AS. Mice with cardiac specific ablation of Yme1l (cYKO), characterized by fragmented mitochondria, showed an increased mortality upon AS challenge. AAV9-hß3AR injection in these mice before AS induction reverted the fragmented mitochondria phenotype and rescued them from death. In conclusion, our results step out that ß3AR overexpression might have translational potential as a therapeutic strategy in AS-induced HF.

GRK2 regulates ADP signaling in platelets via P2Y1 and P2Y12.

The critical role of G protein-coupled receptor kinase 2 (GRK2) in regulating cardiac function has been well documented for >3 decades. Targeting GRK2 has therefore been extensively studied as a novel approach to treating cardiovascular disease. However, little is known about its role in hemostasis and thrombosis. We provide here the first evidence that GRK2 limits platelet activation and regulates the hemostatic response to injury. Deletion of GRK2 in mouse platelets causes increased platelet accumulation after laser-induced injury in the cremaster muscle arterioles, shortens tail bleeding time, and enhances thrombosis in adenosine 5'-diphosphate (ADP)-induced pulmonary thromboembolism and in FeCl3-induced carotid injury. GRK2-/- platelets have increased integrin activation, P-selectin exposure, and platelet aggregation in response to ADP stimulation. Furthermore, GRK2-/- platelets retain the ability to aggregate in response to ADP restimulation, indicating that GRK2 contributes to ADP receptor desensitization. Underlying these changes in GRK2-/- platelets is an increase in Ca2+ mobilization, RAS-related protein 1 activation, and Akt phosphorylation stimulated by ADP, as well as an attenuated rise of cyclic adenosine monophosphate levels in response to ADP in the presence of prostaglandin I2. P2Y12 antagonist treatment eliminates the phenotypic difference in platelet accumulation between wild-type and GRK2-/- mice at the site of injury. Pharmacologic inhibition of GRK2 activity in human platelets increases platelet activation in response to ADP. Finally, we show that GRK2 binds to endogenous Gßγ subunits during platelet activation. Collectively, these results show that GRK2 regulates ADP signaling via P2Y1 and P2Y12, interacts with Gßγ, and functions as a signaling hub in platelets for modulating the hemostatic response to injury.

Metabolic Reprogramming in HIV-Associated Neurocognitive Disorders.

A significant number of patients infected with HIV-1 suffer from HIV-associated neurocognitive disorders (HAND) such as spatial memory impairments and learning disabilities (SMI-LD). SMI-LD is also observed in patients using combination antiretroviral therapy (cART). Our lab has demonstrated that the HIV-1 protein, gp120, promotes SMI-LD by altering mitochondrial functions and energy production. We have investigated cellular processes upstream of the mitochondrial functions and discovered that gp120 causes metabolic reprogramming. Effectively, the addition of gp120 protein to neuronal cells disrupted the glycolysis pathway at the pyruvate level. Looking for the players involved, we found that gp120 promotes increased expression of polypyrimidine tract binding protein 1 (PTBP1), causing the splicing of pyruvate kinase M (PKM) into PKM1 and PKM2. We have also shown that these events lead to the accumulation of advanced glycation end products (AGEs) and prevent the cleavage of pro-brain-derived neurotrophic factor (pro-BDNF) protein into mature brain-derived neurotrophic factor (BDNF). The accumulation of proBDNF results in signaling that increases the expression of the inducible cAMP early repressor (ICER) protein which then occupies the cAMP response element (CRE)-binding sites within the BDNF promoters II and IV, thus altering normal synaptic plasticity. We reversed these events by adding Tepp-46, which stabilizes the tetrameric form of PKM2. Therefore, we concluded that gp120 reprograms cellular metabolism, causing changes linked to disrupted memory in HIV-infected patients and that preventing the disruption of the metabolism presents a potential cure against HAND progression.

Enhanced NCLX-dependent mitochondrial Ca2+ efflux attenuates pathological remodeling in heart failure.

Mitochondrial calcium (mCa2+) uptake couples changes in cardiomyocyte energetic demand to mitochondrial ATP production. However, excessive mCa2+ uptake triggers permeability transition and necrosis. Despite these established roles during acute stress, the involvement of mCa2+ signaling in cardiac adaptations to chronic stress remains poorly defined. Changes in NCLX expression are reported in heart failure (HF) patients and models of cardiac hypertrophy. Therefore, we hypothesized that altered mCa2+ homeostasis contributes to the hypertrophic remodeling of the myocardium that occurs upon a sustained increase in cardiac workload. The impact of mCa2+ flux on cardiac function and remodeling was examined by subjecting mice with cardiomyocyte-specific overexpression (OE) of the mitochondrial Na+/Ca2+ exchanger (NCLX), the primary mediator of mCa2+ efflux, to several well-established models of hypertrophic and non-ischemic HF. Cardiomyocyte NCLX-OE preserved contractile function, prevented hypertrophy and fibrosis, and attenuated maladaptive gene programs in mice subjected to chronic pressure overload. Hypertrophy was attenuated in NCLX-OE mice, prior to any decline in cardiac contractility. NCLX-OE similarly attenuated deleterious cardiac remodeling in mice subjected to chronic neurohormonal stimulation. However, cardiomyocyte NCLX-OE unexpectedly reduced overall survival in mice subjected to severe neurohormonal stress with angiotensin II + phenylephrine. Adenoviral NCLX expression limited mCa2+ accumulation, oxidative metabolism, and de novo protein synthesis during hypertrophic stimulation of cardiomyocytes in vitro. Our findings provide genetic evidence for the contribution of mCa2+ to early pathological remodeling in non-ischemic heart disease, but also highlight a deleterious consequence of increasing mCa2+ efflux when the heart is subjected to extreme, sustained neurohormonal stress.

Epidermal growth factor receptor-dependent maintenance of cardiac contractility.

AIMS: Epidermal growth factor receptor (EGFR) is essential to the development of multiple tissues and organs and is a target of cancer therapeutics. Due to the embryonic lethality of global EGFR deletion and conflicting reports of cardiac-overexpressed EGFR mutants, its specific impact on the adult heart, normally or in response to chronic stress, has not been established. Using complimentary genetic strategies to modulate cardiomyocyte-specific EGFR expression, we aim to define its role in the regulation of cardiac function and remodelling. METHODS AND RESULTS: A floxed EGFR mouse model with α-myosin heavy chain-Cre-mediated cardiomyocyte-specific EGFR downregulation (CM-EGFR-KD mice) developed contractile dysfunction by 9 weeks of age, marked by impaired diastolic relaxation, as monitored via echocardiographic, haemodynamic, and isolated cardiomyocyte contractility analyses. This contractile defect was maintained over time without overt cardiac remodelling until 10 months of age, after which the mice ultimately developed severe heart failure and reduced lifespan. Acute downregulation of EGFR in adult floxed EGFR mice with adeno-associated virus 9 (AAV9)-encoded Cre with a cardiac troponin T promoter (AAV9-cTnT-Cre) recapitulated the CM-EGFR-KD phenotype, while AAV9-cTnT-EGFR treatment of adult CM-EGFR-KD mice rescued the phenotype. Notably, chronic administration of the ß-adrenergic receptor agonist isoproterenol effectively and reversibly compensated for the contractile dysfunction in the absence of cardiomyocyte hypertrophy in CM-EGFR-KD mice. Mechanistically, EGFR downregulation reduced the expression of protein phosphatase 2A regulatory subunit Ppp2r3a/PR72, which was associated with decreased phosphorylation of phospholamban and Ca2+ clearance, and whose re-expression via AAV9-cTnT-PR72 rescued the CM-EGFR-KD phenotype. CONCLUSIONS: Altogether, our study highlights a previously unrecognized role for EGFR in maintaining contractile homeostasis under physiologic conditions in the adult heart via regulation of PR72 expression.

Cardiomyocyte-GSK-3ß deficiency induces cardiac progenitor cell proliferation in the ischemic heart through paracrine mechanisms.

Cardiomyopathy is an irreparable loss and novel strategies are needed to induce resident cardiac progenitor cell (CPC) proliferation in situ to enhance the possibility of cardiac regeneration. Here, we sought to identify the potential roles of glycogen synthase kinase-3ß (GSK-3ß), a critical regulator of cell proliferation and differentiation, in CPC proliferation post-myocardial infarction (MI). Cardiomyocyte-specific conditional GSK-3ß knockout (cKO) and littermate control mice were employed and challenged with MI. Though cardiac left ventricular chamber dimension and contractile functions were comparable at 2 weeks post-MI, cKO mice displayed significantly preserved LV chamber and contractile function versus control mice at 4 weeks post-MI. Consistent with protective phenotypes, an increased percentage of c-kit-positive cells (KPCs) were observed in the cKO hearts at 4 and 6 weeks post-MI which was accompanied by increased levels of cardiomyocyte proliferation. Further analysis revealed that the observed increased number of KPCs in the ischemic cKO hearts was mainly from a cardiac lineage, as the majority of identified KPCs were negative for the hematopoietic lineage marker, CD45. Mechanistically, cardiomyocyte-GSK-3ß profoundly suppresses the expression and secretion of growth factors, including basic-fibroblast growth factor, angiopoietin-2, erythropoietin, stem cell factor, platelet-derived growth factor-BB, granulocyte colony-stimulating factor, and vascular endothelial growth factor, post-hypoxia. In conclusion, our findings strongly suggest that loss of cardiomyocyte-GSK-3ß promotes cardiomyocyte and resident CPC proliferation post-MI. The induction of cardiomyocyte and CPC proliferation in the ischemic cKO hearts is potentially regulated by autocrine and paracrine signaling governed by dysregulated growth factors post-MI. A strategy to inhibit cardiomyocyte-GSK-3ß could be helpful for the promotion of in situ cardiac regeneration post-ischemic injury.

LIN28a induced metabolic and redox regulation promotes cardiac cell survival in the heart after ischemic injury.

RATIONALE: Cell-based therapeutics have been extensively used for cardiac repair yet underperform due to inability of the donated cells to survive in near anoxia after cardiac injury. Cellular metabolism is linked to maintenance of cardiac stem cell (CSC) renewal, proliferation and survival. Ex vivo expansion alters (CSC) metabolism increasing reliance on oxygen dependent respiration. Whether promoting 'metabolic flexibility' in CSCs augments their ability to survive in near anoxia and repair the heart after injury remains untested. OBJECTIVE: Determine the effect of LIN28a induced metabolic flexibility on cardiac tissue derived stem like cell (CTSC) survival and repair after cardiac injury. METHODS AND RESULTS: LIN28a expression coincides during heart development but is lost in adult CTSCs. Reintroduction of LIN28a in adult CTSC (CTSC-LIN) increased proliferation, survival, expression of pluripotency genes and reduced senescence compared to control (CTSC-GFP). Metabolomic analysis show glycolytic intermediates upregulated in CTSC-LIN together with increased lactate production, pyruvate kinase activity, glucose uptake, ECAR and expression of glycolytic enzymes compared to CTSC-GFP. Additionally, CTSC-LIN showed significantly reduced ROS generation and increase antioxidant markers. In response to H2O2 induced oxidative stress, CTSC-LIN showed increased survival and expression of glycolytic genes. LIN28a salutary effects on CTSCs were linked to PDK1/let-7 signaling pathway with loss of PDK1 or alteration of let-7 abrogating LIN28a effects. Following transplantation in the heart after myocardial infarction (MI), CTSC-LIN showed 6% survival rate at day 7 after injection compared to control cells together with increased proliferation and significant increase in cardiac structure and function 8 weeks after MI. Finally, CSTC-LIN showed enhanced ability to secrete paracrine factors under hypoxic conditions and ability to promote cardiomyocyte proliferation following ischemic cardiac injury. CONCLUSIONS: LIN28a modification promotes metabolic flexibility in CTSCs enhancing proliferation and survival post transplantation including ability to repair the heart after myocardial injury.

Identification of a CTRP9 C-Terminal polypeptide capable of enhancing bone-derived mesenchymal stem cell cardioprotection through promoting angiogenic exosome production.

BACKGROUND: Mesenchymal stem cell therapy improves ischemic heart failure via incompletely understood mechanisms. C1q-TNFα related protein-9 (CTRP9) is a novel anti-oxidative cardiokine capable of improving the local microenvironment and cell survival by its c-terminal active globular domain (gCTRP9). The current study attempted to: 1) identify active gCTRP9 c-terminal polypeptides with stem cell protective function; 2) determine whether a lead polypeptide may enable/enhance cortical bone-derived mesenchymal stem cell (CBSC) cardioprotection against post-myocardial infarction (post-MI) remodeling; and 3) define the responsible underlying cellular/molecular mechanisms. METHODS AND RESULTS: Utilizing I-TASSER structure prediction and 3-D active site modeling, we cloned and purified 3 gCTRP9 fragments (CTRP9-237, CTRP9-277, and CTRP9-281). Their activation of cell salvage kinase was compared against gCTRP9. Among the three fragments, CTRP9-281 (a 45 residue-containing polypeptide) exerted comparable or greater ERK1/2 activation compared to gCTRP9. Treatment with CTRP9-281 or gCTRP9 significantly increased CBSC proliferation and migration, and attenuated oxidative stress-induced CBSC apoptosis. CTRP9-281 and gCTRP9 comparably upregulated SOD2 and SOD3 expression. However, CTRP9-281, not gCTRP9, upregulated FGF2 and VEGFA expression/secretion in an ERK1/2 dependent manner. Administration of gCTRP9 or CTRP9-281 alone attenuated post-MI cardiac dysfunction and improved CBSC retention in the infarcted heart in similar fashion. However, CTRP9-281 exerted greater synergistic effect with CBSC than gCTRP9 related to pro-angiogenic, anti-fibrotic, and anti-remodeling effects. Mechanistically, CTRP9-281 significantly increased SOD2-rich and VEGFA-rich exosome production by CBSC. Exosomes from CTRP9-281 treated CBSC significantly attenuated oxidative stress-induced cardiomyocyte apoptosis in vitro. An exosome generation inhibitor attenuated CTRP9-281 enhancement of CBSC cardioprotection in vivo. CONCLUSION: We identified a CTRP9 polypeptide that upregulates SOD2/SOD3 expression and improves CBSC survival/retention, similar to gCTRP9. Moreover, CTRP9-281 stimulates VEGFA-rich exosome production by CBSC, exerting superior pro-angiogenic, anti-fibrotic, and cardioprotective actions.

Targeting GRK5 for Treating Chronic Degenerative Diseases.

G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors and they are responsible for the transduction of extracellular signals, regulating almost all aspects of mammalian physiology. These receptors are specifically regulated by a family of serine/threonine kinases, called GPCR kinases (GRKs). Given the biological role of GPCRs, it is not surprising that GRKs are also involved in several pathophysiological processes. Particular importance is emerging for GRK5, which is a multifunctional protein, expressed in different cell types, and it has been found located in single or multiple subcellular compartments. For instance, when anchored to the plasma membrane, GRK5 exerts its canonical function, regulating GPCRs. However, under certain conditions (e.g., pro-hypertrophic stimuli), GRK5 translocates to the nucleus of cells where it can interact with non-GPCR-related proteins as well as DNA itself to promote "non-canonical" signaling, including gene transcription. Importantly, due to these actions, several studies have demonstrated that GRK5 has a pivotal role in the pathogenesis of chronic-degenerative disorders. This is true in the cardiac cells, tumor cells, and neurons. For this reason, in this review article, we will inform the readers of the most recent evidence that supports the importance of targeting GRK5 to prevent the development or progression of cancer, cardiovascular, and neurological diseases.

KLF5 Is Induced by FOXO1 and Causes Oxidative Stress and Diabetic Cardiomyopathy.

RATIONALE: Diabetic cardiomyopathy (DbCM) is a major complication in type-1 diabetes, accompanied by altered cardiac energetics, impaired mitochondrial function, and oxidative stress. Previous studies indicate that type-1 diabetes is associated with increased cardiac expression of KLF5 (Krüppel-like factor-5) and PPARα (peroxisome proliferator-activated receptor) that regulate cardiac lipid metabolism. OBJECTIVE: In this study, we investigated the involvement of KLF5 in DbCM and its transcriptional regulation. METHODS AND RESULTS: KLF5 mRNA levels were assessed in isolated cardiomyocytes from cardiovascular patients with diabetes and were higher compared with nondiabetic individuals. Analyses in human cells and diabetic mice with cardiomyocyte-specific FOXO1 (Forkhead box protein O1) deletion showed that FOXO1 bound directly on the KLF5 promoter and increased KLF5 expression. Diabetic mice with cardiomyocyte-specific FOXO1 deletion had lower cardiac KLF5 expression and were protected from DbCM. Genetic, pharmacological gain and loss of KLF5 function approaches and AAV (adeno-associated virus)-mediated Klf5 delivery in mice showed that KLF5 induces DbCM. Accordingly, the protective effect of cardiomyocyte FOXO1 ablation in DbCM was abolished when KLF5 expression was rescued. Similarly, constitutive cardiomyocyte-specific KLF5 overexpression caused cardiac dysfunction. KLF5 caused oxidative stress via direct binding on NADPH oxidase (NOX)4 promoter and induction of NOX4 (NADPH oxidase 4) expression. This was accompanied by accumulation of cardiac ceramides. Pharmacological or genetic KLF5 inhibition alleviated superoxide formation, prevented ceramide accumulation, and improved cardiac function in diabetic mice. CONCLUSIONS: Diabetes-mediated activation of cardiomyocyte FOXO1 increases KLF5 expression, which stimulates NOX4 expression, ceramide accumulation, and causes DbCM.

A peptide of the amino-terminus of GRK2 induces hypertrophy and yet elicits cardioprotection after pressure overload.

G protein-coupled receptor (GPCR) kinase 2 (GRK2) expression and activity are elevated early on in response to several forms of cardiovascular stress and are a hallmark of heart failure. Interestingly, though, in addition to its well-characterized role in regulating GPCRs, mounting evidence suggests a GRK2 "interactome" that underlies a great diversity in its functional roles. Several such GRK2 interacting partners are important for adaptive and maladaptive myocyte growth; therefore, an understanding of domain-specific interactions with signaling and regulatory molecules could lead to novel targets for heart failure therapy. Herein, we subjected transgenic mice with cardiac restricted expression of a short, amino terminal fragment of GRK2 (ßARKnt) to pressure overload and found that unlike their littermate controls or previous GRK2 fragments, they exhibited an increased left ventricular wall thickness and mass prior to cardiac stress that underwent proportional hypertrophic growth to controls after acute pressure overload. Importantly, despite this enlarged heart, ßARKnt mice did not undergo the expected transition to heart failure observed in controls. Further, ßARKnt expression limited adverse left ventricular remodeling and increased cell survival signaling. Proteomic analysis to identify ßARKnt binding partners that may underlie the improved cardiovascular phenotype uncovered a selective functional interaction of both endogenous GRK2 and ßARKnt with AKT substrate of 160 kDa (AS160). AS160 has emerged as a key downstream regulator of insulin signaling, integrating physiological and metabolic cues to couple energy demand to membrane recruitment of Glut4. Our preliminary data indicate that in ßARKnt mice, cardiomyocyte insulin signaling is improved during stress, with a coordinate increase in spare respiratory activity and ATP production without metabolite switching. Surprisingly, these studies also revealed a significant decrease in gonadal fat weight, equivalent to human abdominal fat, in male ßARKnt mice at baseline and following cardiac stress. These data suggest that the enhanced AS160-mediated signaling in the ßARKnt mice may ameliorate pathological cardiac remodeling through direct modulation of insulin signaling within cardiomyocytes, and translate these to beneficial effects on systemic metabolism.

Characterization of ßARKct engineered cellular extracellular vesicles and model specific cardioprotection.

Recent data supporting any benefit of stem cell therapy for ischemic heart disease have suggested paracrine-based mechanisms via extracellular vesicles (EVs) including exosomes. We have previously engineered cardiac-derived progenitor cells (CDCs) to express a peptide inhibitor, ßARKct, of G protein-coupled receptor kinase 2, leading to improvements in cell proliferation, survival, and metabolism. In this study, we tested whether ßARKct-CDC EVs would be efficacious when applied to stressed myocytes in vitro and in vivo. When isolated EVs from ßARKct-CDCs and control GFP-CDCs were added to cardiomyocytes in culture, they both protected against hypoxia-induced apoptosis. We tested whether these EVs could protect the mouse heart in vivo, following exposure either to myocardial infarction (MI) or acute catecholamine toxicity. Both types of EVs significantly protected against ischemic injury and improved cardiac function after MI compared with mice treated with EVs from mouse embryonic fibroblasts; however, ßARKct EVs treated mice did display some unique beneficial properties including significantly altered pro- and anti-inflammatory cytokines. Importantly, in a catecholamine toxicity model of heart failure (HF), myocardial injections of ßARKct-containing EVs were superior at preventing HF compared with control EVs, and this catecholamine toxicity protection was recapitulated in vitro. Therefore, introduction of the ßARKct into cellular EVs can have improved reparative properties in the heart especially against catecholamine damage, which is significant as sympathetic nervous system activity is increased in HF.NEW & NOTEWORTHY ßARKct, the peptide inhibitor of GRK2, improves survival and metabolic functions of cardiac-derived progenitor cells. As any benefit of stem cells in the ischemic and injured heart suggests paracrine mechanisms via secreted EVs, we investigated whether CDC-ßARKct engineered EVs would show any benefit over control CDC-EVs. Compared with control EVs, ßARKct-containing EVs displayed some unique beneficial properties that may be due to altered pro- and anti-inflammatory cytokines within the vesicles.

Nitric Oxide and S-Nitrosylation in Cardiac Regulation: G Protein-Coupled Receptor Kinase-2 and ß-Arrestins as Targets.

Cardiac diseases including heart failure (HF), are the leading cause of morbidity and mortality globally. Among the prominent characteristics of HF is the loss of ß-adrenoceptor (AR)-mediated inotropic reserve. This is primarily due to the derangements in myocardial regulatory signaling proteins, G protein-coupled receptor (GPCR) kinases (GRKs) and ß-arrestins (ß-Arr) that modulate ß-AR signal termination via receptor desensitization and downregulation. GRK2 and ß-Arr2 activities are elevated in the heart after injury/stress and participate in HF through receptor inactivation. These GPCR regulators are modulated profoundly by nitric oxide (NO) produced by NO synthase (NOS) enzymes through S-nitrosylation due to receptor-coupled NO generation. S-nitrosylation, which is NO-mediated modification of protein cysteine residues to generate an S-nitrosothiol (SNO), mediates many effects of NO independently from its canonical guanylyl cyclase/cGMP/protein kinase G signaling. Herein, we review the knowledge on the NO system in the heart and S-nitrosylation-dependent modifications of myocardial GPCR signaling components GRKs and ß-Arrs.

MAP Kinase Phosphatase-5 Deficiency Protects Against Pressure Overload-Induced Cardiac Fibrosis.

Cardiac fibrosis, a pathological condition due to excessive extracellular matrix (ECM) deposition in the myocardium, is associated with nearly all forms of heart disease. The processes and mechanisms that regulate cardiac fibrosis are not fully understood. In response to cardiac injury, macrophages undergo marked phenotypic and functional changes and act as crucial regulators of myocardial fibrotic remodeling. Here we show that the mitogen-activated protein kinase (MAPK) phosphatase-5 (MKP-5) in macrophages is involved in pressure overload-induced cardiac fibrosis. Cardiac pressure overload resulting from transverse aortic constriction (TAC) leads to the upregulation of Mkp-5 gene expression in the heart. In mice lacking MKP-5, p38 MAPK and JNK were hyperactivated in the heart, and TAC-induced cardiac hypertrophy and myocardial fibrosis were attenuated. MKP-5 deficiency upregulated the expression of the ECM-degrading matrix metalloproteinase-9 (Mmp-9) in the Ly6Clow (M2-type) cardiac macrophage subset. Consistent with in vivo findings, MKP-5 deficiency promoted MMP-9 expression and activity of pro-fibrotic macrophages in response to IL-4 stimulation. Furthermore, using pharmacological inhibitors against p38 MAPK, JNK, and ERK, we demonstrated that MKP-5 suppresses MMP-9 expression through a combined effect of p38 MAPK/JNK/ERK, which subsequently contributes to the inhibition of ECM-degrading activity. Taken together, our study indicates that pressure overload induces MKP-5 expression and facilitates cardiac hypertrophy and fibrosis. MKP-5 deficiency attenuates cardiac fibrosis through MAPK-mediated regulation of MMP-9 expression in Ly6Clow cardiac macrophages.