Platelets Induce Cell Apoptosis of Cardiac Cells via FasL after Acute Myocardial Infarction.

Acute myocardial infarction (AMI) is one of the leading causes of death worldwide. Cell apoptosis in the myocardium plays an important role in ischemia and reperfusion (I/R) injury, leading to cardiac damage and dysfunction. Platelets are major players in hemostasis and play a crucial role in vessel occlusion, inflammation, and cardiac remodeling after I/R. Here, we studied the impact of platelets on cell apoptosis in the myocardium using a close-chest mouse model of AMI. We found caspase-3-positive resident cardiac cells, while leukocytes were negative for caspase-3. Using two different mouse models of thrombocytopenia, we detected a significant reduction in caspase-3 positive cells in the infarct border zone after I/R injury. Further, we identified platelet FasL to induce cell apoptosis via the extrinsic pathway of Fas receptor activation of target cells. Mechanistically, hypoxia triggers platelet adhesion to FasR, suggesting that platelet-induced apoptosis is elevated after I/R. Platelet-specific FasL knock-out mice showed reduced Bax and Bcl2 expression, suggesting that platelets modulate the intrinsic and extrinsic pathways of apoptosis, leading to reduced infarct size after myocardial I/R injury. Thus, a new mechanism for how platelets contribute to tissue homeostasis after AMI was identified that should be validated in patients soon.

Ischemia does not provoke the full immune training repertoire in human cardiac fibroblasts.

Trained immunity of monocytes, endothelial, and smooth muscle cells augments the cytokine response to secondary stimuli. Immune training is characterized by stabilization of hypoxia-inducible factor (HIF)-1α, mTOR activation, and aerobic glycolysis. Cardiac fibroblast (CF)-myofibroblast transition upon myocardial ischemia/reperfusion (I/R) features epigenetic and metabolic adaptations reminiscent of trained immunity. We assessed the impact of I/R on characteristics of immune training in human CF and mouse myocardium. I/R was simulated in vitro with transient metabolic inhibition. CF primed with simulated I/R or control buffer were 5 days later re-stimulated with Pam3CSK for 24 h. Mice underwent transient left anterior descending artery occlusion or sham operation with reperfusion for up to 5 days. HIF-regulated metabolic targets and cytokines were assessed by qPCR, immunoblot, and ELISA and glucose consumption, lactate release, and lactate dehydrogenase (LDH) by chromogenic assay. Simulated I/R increased HIF-1α stabilization, mTOR phosphorylation, glucose consumption, lactate production, and transcription of PFKB3 and F2RL3, a HIF-regulated target gene, in human CF. PGK1 and LDH mRNAs were suppressed. Intracellular LDH transiently increased after simulated I/R, and extracellular LDH showed sustained elevation. I/R priming increased abundance of pro-caspase-1, auto-cleaved active caspase-1, and the expression and secretion of interleukin (IL)-1ß, but did not augment Pam3CSK-stimulated cytokine transcription or secretion. Myocardial I/R in vivo increased abundance of HIF-1 and the precursor and cleaved forms of caspase-1, caspase-11, and caspase-8, but not of LDH-A or phospho-mTOR. I/R partially reproduces features of immune training in human CF, specifically HIF-1α stabilization, aerobic glycolysis, mTOR phosphorylation, and PFKB3 transcription. I/R does not augment PGK1 or LDH expression or the cytokine response to Pam3CSK. Regulation of PAR4 and inflammasome caspases likely occurs independently of an immune training repertoire.

Constitutive Activation of gp130 in T Cells Results in Senescence and Premature Aging.

IL-6 family members contribute to host defense through the stimulation of acute-phase signaling, hematopoiesis, immune reactions, and regenerative processes. To investigate essential mechanisms that are linked toward a constitutively activated gp130 signaling, we generated and characterized a mouse model that reflects a constitutive and cytokine-independent activation of JAK/STAT3 signaling by Lgp130 in CD4- and CD8-positive T cells. Lgp130 is an engineered form of gp130 in which dimerization and activation are forced by a leucine zipper. T cell-specific Lgp130 activation resulted in massive phenotypical abnormalities, including splenomegaly, lymphadenopathy, and an upregulation of innate immune system components shown by hyperinflammatory signatures in several organs. Moreover, T cell-restricted expression of Lgp130 resulted in increased numbers of cytotoxic and regulatory T cells, especially in lymph nodes. Consistent with this, we found an elevated platelet production and increase in megakaryocytes in the spleen and bone marrow that are causative for an acute thrombocytosis accompanied by anemia. Due to a shortened life span of T cell-specific Lgp130 mice, we could also show that next to an overall increase in regulatory cell-cycle genes, an activation of p53 and increased expression of p21 provide evidence for a senescence-like phenotype. Together, these data suggest that T cell-restricted gp130 activation is not only involved in autoimmune processes but also in senescence-associated aging. Therefore, Lgp130 expression in T cells might be a suitable model to study inflammation and disease.

Cardiac ischemia modulates white adipose tissue in a depot-specific manner.

The incidence of heart failure after myocardial infarction (MI) remains high and the underlying causes are incompletely understood. The crosstalk between heart and adipose tissue and stimulated lipolysis has been identified as potential driver of heart failure. Lipolysis is also activated acutely in response to MI. However, the role in the post-ischemic remodeling process and the contribution of different depots of adipose tissue is unclear. Here, we employ a mouse model of 60 min cardiac ischemia and reperfusion (I/R) to monitor morphology, cellular infiltrates and gene expression of visceral and subcutaneous white adipose tissue depots (VAT and SAT) for up to 28 days post ischemia. We found that in SAT but not VAT, adipocyte size gradually decreased over the course of reperfusion and that these changes were associated with upregulation of UCP1 protein, indicating white adipocyte conversion to the so-called 'brown-in-white' phenotype. While this phenomenon is generally associated with beneficial metabolic consequences, its role in the context of MI is unknown. We further measured decreased lipogenesis in SAT together with enhanced infiltration of MAC-2+ macrophages. Finally, quantitative PCR analysis revealed transient downregulation of the adipokines adiponectin, leptin and resistin in SAT. While adiponectin and leptin have been shown to be cardioprotective, the role of resistin after MI needs further investigation. Importantly, all significant changes were identified in SAT, while VAT was largely unaffected by MI. We conclude that targeted interference with lipolysis in SAT may be a promising approach to promote cardiac healing after ischemia.

Only Acute but Not Chronic Thrombocytopenia Protects Mice against Left Ventricular Dysfunction after Acute Myocardial Infarction.

BACKGROUND: Platelets are major players of thrombosis and inflammation after acute myocardial infarction (AMI). The impact of thrombocytopenia on platelet-induced cellular processes post AMI is not well defined. METHODS: The left anterior descending artery was ligated in C57/Bl6 mice and in two thrombocytopenic mouse models to induce AMI. RESULTS: Platelets from STEMI patients and from C57/Bl6 mice displayed enhanced platelet activation after AMI. This allows platelets to migrate into the infarct but not into the remote zone of the left ventricle. Acute thrombocytopenia by antibody-induced platelet depletion resulted in reduced infarct size and improved cardiac function 24 h and 21 days post AMI. This was due to reduced platelet-mediated inflammation after 24 h and reduced scar formation after 21 days post AMI. The collagen composition and interstitial collagen content in the left ventricle were altered due to platelet interaction with cardiac fibroblasts. Acute inflammation was also significantly reduced in Mpl-/- mice with chronic thrombocytopenia, but cardiac remodeling was unaltered. Consequently, left ventricular function, infarct size and scar formation in Mpl-/- mice were comparable to controls. CONCLUSION: This study discovers a novel role for platelets in cardiac remodeling and reveals that acute but not chronic thrombocytopenia protects left ventricular function post AMI.

Diabetes disturbs functional adaptation of the remote myocardium after ischemia/reperfusion.

Diabetes mellitus type 2 is associated with adverse clinical outcome after myocardial infarction. To better understand the underlying causes we here investigated sarcomere protein function and its calcium-dependent regulation in the non-ischemic remote myocardium (RM) of diabetic mice (db/db) after transient occlusion of the left anterior descending coronary artery. Before and 24 h after surgery db/db and non-diabetic db/+ underwent magnetic resonance imaging followed by histological and biochemical analyses of heart tissue. Intracellular calcium transients and sarcomere function were measured in isolated cardiomyocytes. Active and passive force generation was assessed in skinned fibers and papillary muscle preparations. Before ischemia and reperfusion (I/R), beat-to-beat calcium cycling was depressed in diabetic cardiomyocytes. Nevertheless, contractile function was preserved owing to increased myofilament calcium sensitivity and higher responsiveness of myocardial force production to ß-adrenergic stimulation in db/db compared to db/+. In addition, protein kinase C activity was elevated in db/db hearts leading to strong phosphorylation of the titin PEVK region and increased titin-based tension of myofilaments. I/R impaired the function of whole hearts and RM sarcomeres in db/db to a larger extent than in non-diabetic db/+, and we identified several reasons. First, the amplitude and the kinetics of cardiomyocyte calcium transients were further reduced in the RM of db/db. Underlying causes involved altered expression of calcium regulatory proteins. Diabetes and I/R additively reduced phospholamban S16-phosphorylation by 80% (P < 000.1) leading to strong inhibition of the calcium ATPase SERCA2a. Second, titin stiffening was only observed in the RM of db/+, but not in the RM of db/db. Finally, db/db myofilament calcium sensitivity and force generation upon ß-adrenergic stimulation were no longer enhanced over db/+ in the RM. The findings demonstrate that impaired cardiomyocyte calcium cycling of db/db hearts is compensated by increased myofilament calcium sensitivity and increased titin-based stiffness prior to I/R. In contrast, sarcomere function of the RM 24 h after I/R is poor because both these compensatory mechanisms fail and myocyte calcium handling is further depressed.

Hyaluronan synthase 3 is protective after cardiac ischemia-reperfusion by preserving the T cell response.

Dysregulated extracellular matrix (ECM) is a hallmark of adverse cardiac remodeling after myocardial infarction (MI). Previous work from our laboratory suggests that synthesis of the major ECM component hyaluronan (HA) may be beneficial for post-infarct healing. Here, we aimed to investigate the mechanisms of hyaluronan synthase 3 (HAS3) in cardiac healing after MI. Mice with genetic deletion of Has3 (Has3 KO) and wildtype mice (WT) underwent 45 min of ischemia with subsequent reperfusion (I/R), followed by monitoring of heart function and analysis of tissue remodeling for up to three weeks. Has3 KO mice exhibited impaired cardiac function as evidenced by a reduced ejection fraction. Accordingly, Has3 deficiency also resulted in an increased scar size. Cardiac fibroblast activation and CD68+ macrophage counts were similar between genotypes. However, we found a significant decrease in CD4 T cells in the hearts of Has3 KO mice seven days post-MI, in particular reduced numbers of CD4+CXCR3+ Th1 and CD4+CD25+Treg cells. Furthermore, Has3 deficient cardiac T cells were less activated and more apoptotic as shown by decreased CD69+ and increased annexin V+ cells, respectively. In vitro assays using activated splenic CD3 T cells demonstrated that Has3 deficiency resulted in reduced expression of the main HA receptor CD44 and diminished T cell proliferation. T cell transendothelial migration was similar between genotypes. Of note, analysis of peripheral blood from patients with ST-elevation myocardial infarction (STEMI) revealed that HAS3 is the predominant HAS isoenzyme also in human T cells. In conclusion, our data suggest that HAS3 is required for mounting a physiological T cell response after MI to support cardiac healing. Therefore, our study may serve as a foundation for the development of novel strategies targeting HA-matrix to preserve T cell function after MI.

4-Methylumbelliferone Attenuates Macrophage Invasion and Myocardial Remodeling in Pressure-Overloaded Mouse Hearts.

[Figure: see text].

Enhanced Integrin Activation of PLD2-Deficient Platelets Accelerates Inflammation after Myocardial Infarction.

BACKGROUND: Phospholipase (PL)D1 is crucial for integrin αIIbß3 activation of platelets in arterial thrombosis and TNF-α-mediated inflammation and TGF-ß-mediated collagen scar formation after myocardial infarction (MI) in mice. Enzymatic activity of PLD is not responsible for PLD-mediated TNF-α signaling and myocardial healing. The impact of PLD2 in ischemia reperfusion injury is unknown. METHODS: PLD2-deficient mice underwent myocardial ischemia and reperfusion (I/R). RESULTS: Enhanced integrin αIIbß3 activation of platelets resulted in elevated interleukin (IL)-6 release from endothelial cells in vitro and enhanced IL-6 plasma levels after MI in PLD2-deficient mice. This was accompanied by enhanced migration of inflammatory cells into the infarct border zone and reduced TGF-ß plasma levels after 72 h that might account for enhanced inflammation in PLD2-deficient mice. In contrast to PLD1, TNF-α signaling, infarct size and cardiac function 24 h after I/R were not altered when PLD2 was deleted. Furthermore, TGF-ß plasma levels, scar formation and heart function were comparable between PLD2-deficient and control mice 21 days post MI. CONCLUSIONS: The present study contributes to our understanding about the role of PLD isoforms and altered platelet signaling in the process of myocardial I/R injury.

Cardiac Hyaluronan Synthesis Is Critically Involved in the Cardiac Macrophage Response and Promotes Healing After Ischemia Reperfusion Injury.

RATIONALE: Immediate changes in the ECM (extracellular matrix) microenvironment occur after myocardial ischemia and reperfusion (I/R) injury. OBJECTIVE: Aim of this study was to unravel the role of the early hyaluronan (HA)-rich ECM after I/R. METHODS AND RESULTS: Genetic deletion of Has2 and Has1 was used in a murine model of cardiac I/R. Chemical exchange saturation transfer imaging was adapted to image cardiac ECM post-I/R. Of note, the cardiac chemical exchange saturation transfer signal was severely suppressed by Has2 deletion and pharmacological inhibition of HA synthesis 24 hours after I/R. Has2 KO ( Has2 deficient) mice showed impaired hemodynamic function suggesting a protective role for endogenous HA synthesis. In contrast to Has2 deficiency, Has1-deficient mice developed no specific phenotype compared with control post-I/R. Importantly, in Has2 KO mice, cardiac macrophages were diminished after I/R as detected by 19F MRI (magnetic resonance imaging) of perfluorcarbon-labeled immune cells, Mac-2/Galectin-3 immunostaining, and FACS (fluorescence-activated cell sorting) analysis (CD45+CD11b+Ly6G-CD64+F4/80+cells). In contrast to macrophages, cardiac Ly6Chigh and Ly6Clow monocytes were unaffected post-I/R compared with control mice. Mechanistically, inhibition of HA synthesis led to increased macrophage apoptosis in vivo and in vitro. In addition, α-SMA (α-smooth muscle actin)-positive cells were reduced in the infarcted myocardium and in the border zone. In vitro, the myofibroblast response as measured by Acta2 mRNA expression was reduced by inhibition of HA synthesis and of CD44 signaling. Furthermore, Has2 KO fibroblasts were less able to contract collagen gels in vitro. The effects of HA/CD44 on fibroblasts and macrophages post-I/R might also affect intercellular cross talk because cardiac fibroblasts were activated by monocyte/macrophages and, in turn, protected macrophages from apoptosis. CONCLUSIONS: Increased HA synthesis contributes to postinfarct healing by supporting macrophage survival and by promoting the myofibroblast response. Additionally, imaging of cardiac HA by chemical exchange saturation transfer post-I/R might have translational value.

Enzymatic Activity Is Not Required for Phospholipase D Mediated TNF-α Regulation and Myocardial Healing.

Phospholipase D1 is a regulator of tumor necrosis factor-α expression and release upon LPS-induced sepsis and following myocardial infarction (MI). Lack of PLD1 leads to a reduced TNF-α mediated inflammatory response and to enhanced infarct size with declined cardiac function 21 days after ischemia reperfusion (I/R) injury. Deficiency of both PLD isoforms PLD1 and PLD2 as well as pharmacological inhibition of the enzymatic activity of PLD with the PLD inhibitor FIPI protected mice from arterial thrombosis and ischemic brain infarction. Here we treated mice with the PLD inhibitor FIPI to analyze if pharmacological inhibition of PLD after myocardial ischemia protects mice from cardiac damage. Inhibition of PLD with FIPI leads to reduced migration of inflammatory cells into the infarct border zone 24 h after experimental MI in mice, providing first evidence for immune cell migration to be dependent on the enzymatic activity of PLD. In contrast to PLD1 deficient mice, TNF-α plasma level was not altered after FIPI treatment of mice. Consequently, infarct size and left ventricular (LV) function were comparable between FIPI-treated and control mice 21 days post MI. Moreover, cell survival 24 h post I/R was not altered upon FIPI treatment. Our results indicate that the enzymatic activity of PLD is not responsible for PLD mediated TNF-α signaling and myocardial healing after I/R injury in mice. Furthermore, reduced TNF-α plasma levels in PLD1 deficient mice might be responsible for increased infarct size and impaired cardiac function 21 days post MI.

IL-23R Signaling Plays No Role in Myocardial Infarction.

Ischemic heart diseases are the most frequent diseases in the western world. Apart from Interleukin (IL-)1, inflammatory therapeutic targets in the clinic are still missing. Interestingly, opposing roles of the pro-inflammatory cytokine IL-23 have been described in cardiac ischemia in mice. IL-23 is a composite cytokine consisting of p19 and p40 which binds to IL-23R and IL-12Rß1 to initiate signal transduction characterized by activation of the Jak/STAT, PI3K and Ras/Raf/MAPK pathways. Here, we generate IL-23R-Y416FΔICD signaling deficient mice and challenged these mice in close- and open-chest left anterior descending coronary arteria ischemia/reperfusion experiments. Our experiments showed only minimal changes in all assayed parameters in IL-23R signaling deficient mice compared to wild-type mice in ischemia and for up to four weeks of reperfusion, including ejection fraction, endsystolic volume, enddiastolic volume, infarct size, gene regulation and α smooth muscle actin (αSMA) and Hyaluronic acid (HA) protein expression. Moreover, injection of IL-23 in wild-type mice after LAD ischemia/reperfusion had also no influence on the outcome of the healing phase. Our data showed that IL-23R deficiency has no effects in myocardial I/R.

A systems biology network analysis of nutri(epi)genomic changes in endothelial cells exposed to epicatechin metabolites.

Although vasculo-protective effects of flavan-3-ols are widely accepted today, their impact on endothelial cell functions and molecular mechanisms of action involved is not completely understood. The aim of this study was to characterize the potential endothelium-protective effects of circulating epicatechin metabolites and to define underlying mechanisms of action by an integrated systems biology approach. Reduced leukocyte rolling over vascular endothelium was observed following epicatechin supplementation in a mouse model of inflammation. Integrative pathway analysis of transcriptome, miRNome and epigenome profiles of endothelial cells exposed to epicatechin metabolites revealed that by acting at these different levels of regulation, metabolites affect cellular pathways involved in endothelial permeability and interaction with immune cells. In-vitro experiments on endothelial cells confirmed that epicatechin metabolites reduce monocyte adhesion and their transendothelial migration. Altogether, our in-vivo and in-vitro results support the outcome of a systems biology based network analysis which suggests that epicatechin metabolites mediate their vasculoprotective effects through dynamic regulation of endothelial cell monocyte adhesion and permeability. This study illustrates complex and multimodal mechanisms of action by which epicatechin modulate endothelial cell integrity.

CDKN1B/p27 is localized in mitochondria and improves respiration-dependent processes in the cardiovascular system-New mode of action for caffeine.

We show that the cyclin-dependent kinase inhibitor 1B (CDKN1B)/p27, previously known as a cell cycle inhibitor, is also localized within mitochondria. The migratory capacity of endothelial cells, which need intact mitochondria, is completely dependent on mitochondrial p27. Mitochondrial p27 improves mitochondrial membrane potential, increases adenosine triphosphate (ATP) content, and is required for the promigratory effect of caffeine. Domain mapping of p27 revealed that the N-terminus and C-terminus are required for those improvements. Further analysis of those regions revealed that the translocation of p27 into the mitochondria and its promigratory activity depend on serine 10 and threonine 187. In addition, mitochondrial p27 protects cardiomyocytes against apoptosis. Moreover, mitochondrial p27 is necessary and sufficient for cardiac myofibroblast differentiation. In addition, p27 deficiency and aging decrease respiration in heart mitochondria. Caffeine does not increase respiration in p27-deficient animals, whereas aged mice display improvement after 10 days of caffeine in drinking water. Moreover, caffeine induces transcriptome changes in a p27-dependent manner, affecting mostly genes relevant for mitochondrial processes. Caffeine also reduces infarct size after myocardial infarction in prediabetic mice and increases mitochondrial p27. Our data characterize mitochondrial p27 as a common denominator that improves mitochondria-dependent processes and define an increase in mitochondrial p27 as a new mode of action of caffeine.

Impaired Ca2+ cycling of nonischemic myocytes contributes to sarcomere dysfunction early after myocardial infarction.

Changes in the nonischemic remote myocardium of the heart contribute to left ventricular dysfunction after ischemia and reperfusion (I/R). Understanding the underlying mechanisms early after I/R is crucial to improve the adaptation of the viable myocardium to increased mechanical demands. Here, we investigated the role of myocyte Ca2+ handling in the remote myocardium 24 h after 60 min LAD occlusion. Cardiomyocytes isolated from the basal noninfarct-related parts of wild type mouse hearts demonstrated depressed beat-to-beat Ca2+ handling. The amplitude of the Ca2+ transients as well as the kinetics of Ca2+ transport were reduced by up to 25%. These changes were associated with impaired sarcomere contraction. While expression levels of Ca2+ regulatory proteins were unchanged in remote myocardium compared to the corresponding regions of sham-operated hearts, mobility shift analyses of phosphorylated protein showed 2.9 ±â€¯0.4-fold more unphosphorylated phospholamban (PLN) monomers, the PLN species that inhibits the Ca2+ ATPase SERCA2a (P ≤ 0.001). Phospho-specific antibodies revealed normal phosphorylation of PLN at T17 in remote myocardium, but markedly reduced phosphorylation at its PKA-dependent phosphorylation site, S16 (P ≤ 0.01). The underlying cause involved enhanced activity of protein phosphatases, particularly PP2A (P ≤ 0.01). In contrast, overall PKA activity was normal. The PLN interactome, as determined by co-immunoprecipitation and mass spectrometry, and the phosphorylation state of PKA targets other than PLN were also unchanged. Isoproterenol enhanced cellular Ca2+ cycling much stronger in remote myocytes than in healthy controls and improved sarcomere function. We conclude that the reduced phosphorylation state of PLN at S16 impairs myocyte Ca2+ cycling in the remote myocardium 24 h after I/R and contributes to contractile dysfunction.

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video.

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

Titin-Based Cardiac Myocyte Stiffening Contributes to Early Adaptive Ventricular Remodeling After Myocardial Infarction.

RATIONALE: Myocardial infarction (MI) increases the wall stress in the viable myocardium and initiates early adaptive remodeling in the left ventricle to maintain cardiac output. Later remodeling processes include fibrotic reorganization that eventually leads to cardiac failure. Understanding the mechanisms that support cardiac function in the early phase post MI and identifying the processes that initiate transition to maladaptive remodeling are of major clinical interest. OBJECTIVE: To characterize MI-induced changes in titin-based cardiac myocyte stiffness and to elucidate the role of titin in ventricular remodeling of remote myocardium in the early phase after MI. METHODS AND RESULTS: Titin properties were analyzed in Langendorff-perfused mouse hearts after 20-minute ischemia/60-minute reperfusion (I/R), and mouse hearts that underwent ligature of the left anterior descending coronary artery for 3 or 10 days. Cardiac myocyte passive tension was significantly increased 1 hour after ischemia/reperfusion and 3 and 10 days after left anterior descending coronary artery ligature. The increased passive tension was caused by hypophosphorylation of the titin N2-B unique sequence and hyperphosphorylation of the PEVK (titin domain rich in proline, glutamate, valine, and lysine) region of titin. Blocking of interleukine-6 before left anterior descending coronary artery ligature restored titin-based myocyte tension after MI, suggesting that MI-induced titin stiffening is mediated by elevated levels of the cytokine interleukine-6. We further demonstrate that the early remodeling processes 3 days after MI involve accelerated titin turnover by the ubiquitin-proteasome system. CONCLUSIONS: We conclude that titin-based cardiac myocyte stiffening acutely after MI is partly mediated by interleukine-6 and is an important mechanism of remote myocardium to adapt to the increased mechanical demands after myocardial injury.

Oligophrenin1 protects mice against myocardial ischemia and reperfusion injury by modulating inflammation and myocardial apoptosis.

The Rho family of small GTPases has been analyzed in cardiac physiology and pathophysiology including myocardial infarction (MI) in the last years. Contradictory results show either a protective or a declined effect of RhoA and the RhoA effector Rho-associated protein kinase (ROCK) in myocardial ischemia and reperfusion injury that is associated with cardiomyocyte survival and caspase-3 activation. Cardiac-specific deletion of Rac1 reduced ischemia reperfusion injury in diabetic hearts, whereas cardiomyocyte specific overexpression of active Rac1 predisposes the heart to increased myocardial injury with enhanced contractile dysfunction. GTPase-activating proteins (GAPs) control the activation of Rho proteins through stimulation of GTP hydrolysis. However, the impact of GAPs in myocardial ischemia and reperfusion injury remains elusive. Here we analyzed the role of oligophrenin1 (OPHN1), a RhoGAP with Bin/Amphiphysin/Rvs (BAR) domain known to regulate the activity of RhoA, Rac1 and Cdc42 in MI. The expression of Ophn1, RhoA and Rac1 is strongly upregulated 24h after myocardial ischemia. Loss of OPHN1 induced enhanced activity of Rho effector molecules leading to elevated cardiomyocyte apoptosis and increased migration of inflammatory cells into the infarct border zone of OPHN1 deficient mice. Consequently, echocardiography 24h after myocardial ischemia revealed declined left ventricle function in OPHN1 deficient mice. Our results indicate that OPHN1 mediated regulation of RhoA, Rac1 and Cdc42 is crucial for the preservation of cardiac function after myocardial injury.

Circulating NOS3 modulates left ventricular remodeling following reperfused myocardial infarction.

PURPOSE: Nitric oxide (NO) is constitutively produced and released from the endothelium and several blood cell types by the isoform 3 of the NO synthase (NOS3). We have shown that NO protects against myocardial ischemia/reperfusion (I/R) injury and that depletion of circulating NOS3 increases within 24 h of ischemia/reperfusion the size of myocardial infarction (MI) in chimeric mice devoid of circulating NOS3. In the current study we hypothesized that circulating NOS3 also affects remodeling of the left ventricle following reperfused MI. METHODS: To analyze the role of circulating NOS3 we transplanted bone marrow of NOS3-/- and wild type (WT) mice into WT mice, producing chimerae expressing NOS3 only in vascular endothelium (BC-/EC+) or in both, blood cells and vascular endothelium (BC+/EC+). Both groups underwent 60 min of coronary occlusion in a closed-chest model of reperfused MI. During the 3 weeks post MI, structural and functional LV remodeling was serially assessed (24 h, 4 d, 1 w, 2 w and 3 w) by echocardiography. At 72 hours post MI, gene expression of several extracellular matrix (ECM) modifying molecules was determined by quantitative RT-PCR analysis. At 3 weeks post MI, hemodynamics were obtained by pressure catheter, scar size and collagen content were quantified post mortem by Gomori's One-step trichrome staining. RESULTS: Three weeks post MI, LV end-systolic (53.2±5.9 µl; ***p≤0.001; n = 5) and end-diastolic volumes (82.7±5.6 µl; *p<0.05; n = 5) were significantly increased in BC-/EC+, along with decreased LV developed pressure (67.5±1.8 mm Hg; n = 18; ***p≤0.001) and increased scar size/left ventricle (19.5±1.5%; n = 13; **p≤0.01) compared to BC+/EC+ (ESV: 35.6±2.2 µl; EDV: 69.1±2.6 µl n = 8; LVDP: 83.2±3.2 mm Hg; n = 24; scar size/LV13.8±0.7%; n = 16). Myocardial scar of BC-/EC+ was characterized by increased total collagen content (20.2±0.8%; n = 13; ***p≤0.001) compared to BC+/EC+ (15.9±0.5; n = 16), and increased collagen type I and III subtypes. CONCLUSION: Circulating NOS3 ameliorates maladaptive left ventricular remodeling following reperfused myocardial infarction.