IGF Signaling in the Heart in Health and Disease.

One of the most vital processes of the body is the cardiovascular system's proper operation. Physiological processes in the heart are regulated by the balance of cardioprotective and pathological mechanisms. The insulin-like growth factor system (IGF system, IGF signaling pathway) plays a pivotal role in regulating growth and development of various cells and tissues. In myocardium, the IGF system provides cardioprotective effects as well as participates in pathological processes. This review summarizes recent data on the role of IGF signaling in cardioprotection and pathogenesis of various cardiovascular diseases, as well as analyzes severity of these effects in various scenarios.

Specific cleavage of IGFBP-4 by papp-a in nervous tissue.

Astrocytes are subtypes of glial cells involved in metabolic, structural, homeostatic, and neuroprotective processes that help neurons maintain viability. Insulin-like growth factors IGF-1 and IGF-2 are known to have neuroprotective effects on neurons and glial cells through interaction with specific receptors. IGF forms a complex with IGF-binding proteins (IGFBP) in nervous tissue and is released from the complex via IGFBP proteolysis by specific proteases. It has been reported that IGFBP-2, 5 and 6 are cleaved by specific proteases in the central nervous system (CNS), followed by IGF release; however, it was unknown whether IGFBP-4 was exposed to a particular proteolysis in nervous tissue. Using neurons and astrocytes derived from human induced pluripotent stem cell lines (hiPSC), as well as rat brain-sourced primary neuron-glia cultures, we demonstrated that IGFBP-4 is specifically cleaved in nervous tissue by the Pregnancy Associated Plasma Protein A (PAPP-A) protease and that this cleavage is IGF-dependent. Our results indicate that astrocyte rather than neuron PAPP-A cleaves IGFBP-4 in nervous tissue suggesting that this may be one of the fundamental mechanisms for IGF interchange between these two types of cells.

Redox differences between rat neonatal and adult cardiomyocytes under hypoxia.

It is generally accepted that oxidative stress plays a key role in the development of ischemia-reperfusion injury in ischemic heart disease. However, the mechanisms how reactive oxygen species trigger cellular damage are not fully understood. Our study investigates redox state and highly reactive substances within neonatal and adult cardiomyocytes under hypoxia conditions. We have found that hypoxia induced an increase in H2O2 production in adult cardiomyocytes, while neonatal cardiomyocytes experienced a decrease in H2O2 levels. This finding correlates with our observation of the difference between the electron transport chain (ETC) properties and mitochondria amount in adult and neonatal cells. We demonstrated that in adult cardiomyocytes hypoxia caused the significant increase in the ETC loading with electrons compared to normoxia. On the contrary, in neonatal cardiomyocytes ETC loading with electrons was similar under both normoxic and hypoxic conditions that could be due to ETC non-functional state and the absence of the electrons transfer to O2 under normoxia. In addition to the variations in H2O2 production, we also noted consistent pH dynamics under hypoxic conditions. Notably, the pH levels exhibited a similar decrease in both cell types, thus, acidosis is a more universal cellular response to hypoxia. We also demonstrated that the amount of mitochondria and the levels of cardiac isoforms of troponin I, troponin T, myoglobin and GAPDH were significantly higher in adult cardiomyocytes compared to neonatal ones. Remarkably, we found out that under hypoxia, the levels of cardiac isoforms of troponin T, myoglobin, and GAPDH were elevated in adult cardiomyocytes, while their level in neonatal cells remained unchanged. Obtained data contribute to the understanding of the mechanisms of neonatal cardiomyocytes' resistance to hypoxia and the ability to maintain the metabolic homeostasis in contrast to adult ones.

PAPP-A-Specific IGFBP-4 Proteolysis in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

The insulin-like growth factors IGF-I and IGF-II-as well as their binding proteins (IGFBPs), which regulate their bioavailability-are involved in many pathological and physiological processes in cardiac tissue. Pregnancy-associated plasma protein A (PAPP-A) is a metalloprotease that preferentially cleaves IGFBP-4, releasing IGF and activating its biological activity. Previous studies have shown that PAPP-A-specific IGFBP-4 proteolysis is involved in the pathogenesis of cardiovascular diseases, such as ischemia, heart failure, and acute coronary syndrome. However, it remains unclear whether PAPP-A-specific IGFBP-4 proteolysis participates in human normal cardiomyocytes. Here, we report PAPP-A-specific IGFBP-4 proteolysis occurring in human cardiomyocytes derived from two independent induced pluripotent cell lines (hiPSC-CMs), detected both on the cell surface and in the cell secretome. PAPP-A was measured by fluoroimmune analysis (FIA) in a conditioned medium of hiPSC-CMs and was detected in concentrations of up to 4.3 ± 1.33 ng/mL and 3.8 ± 1.1 ng/mL. The level of PAPP-A-specific IGFBP-4 proteolysis was determined as the concentration of NT-IGFBP-4 proteolytic fragments using FIA for a proteolytic neo-epitope-specific assay. We showed that PAPP-A-specific IGFBP-4 proteolysis is IGF-dependent and inhibited by EDTA and 1,10-phenanthroline. Therefore, it may be concluded that PAPP-A-specific IGFBP-4 proteolysis functions in human normal cardiomyocytes, and hiPSC-CMs contain membrane-bound and secreted forms of proteolytically active PAPP-A.

IGF-Binding Proteins and Their Proteolysis as a Mechanism of Regulated IGF Release in the Nervous Tissue.

Insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) play a key role in the maintenance of the nervous tissue viability. IGF-1 and IGF-2 exhibit the neuroprotective effects by stimulating migration and proliferation of nervous cells, activating cellular metabolism, inducing regeneration of damaged cells, and regulating various stages of prenatal and postnatal development of the nervous system. The availability of IGFs for the cells is controlled via their interaction with the IGF-binding proteins (IGFBPs) that inhibit their activity. On the contrary, the cleavage of IGFBPs by specific proteases leads to the IGF release and activation of its cellular effects. The viability of neurons in the nervous tissue is controlled by a complex system of trophic factors secreted by auxiliary glial cells. The main source of IGF for the neurons are astrocytes. IGFs can accumulate as an extracellular free ligand near the neuronal membranes as a result of proteolytic degradation of IGFBPs by proteases secreted by astrocytes. This mechanism promotes interaction of IGFs with their genuine receptors and triggers intracellular signaling cascades. Therefore, the release of IGF by proteolytic cleavage of IGFBPs is an important mechanism of neuronal protection. This review summarizes the published data on the role of IGFs and IGFBPs as the key players in the neuroprotective regulation with a special focus on the specific proteolysis of IGFBPs as a mechanism for the regulation of IGF bioavailability and viability of neurons.

IGFBP-4 Proteolysis by PAPP-A in a Primary Culture of Rat Neonatal Cardiomyocytes under Normal and Hypertrophic Conditions.

Cardiovascular diseases (CVD) are among the leading causes of death and disability worldwide. Pregnancy-associated plasma protein-A (PAPP-A) is a matrix metalloprotease localized on the cell surface. One of the substrates that PAPP-A cleaves is the insulin-like growth factor binding protein-4 (IGFBP-4), a member of the family of proteins that bind insulin-like growth factor (IGF). Proteolysis of IGFBP-4 by PAPP-A occurs at a specific site resulting in formation of two proteolytic fragments - N-terminal IGFBP-4 (NT-IGFBP-4) and C-terminal IGFBP-4 (CT-IGFBP-4), and leads to the release of IGF activating various cellular processes including migration, proliferation, and cell growth. Increased levels of the proteolytic IGFBP-4 fragments correlate with the development of CVD complications and increased risk of death in patients with the coronary heart disease, acute coronary syndrome, and heart failure. However, there is no direct evidence that PAPP-A specifically cleaves IGFBP-4 in the cardiac tissue under normal and pathological conditions. In the present study, using a primary culture of rat neonatal cardiomyocytes as a model, we have demonstrated that: 1) proteolysis of IGFBP-4 by PAPP-A occurs in the conditioned medium of cardiomyocytes, 2) PAPP-A-specific IGFBP-4 proteolysis is increased when cardiomyocytes are transformed to a hypertrophic state. Thus, it can be assumed that the enhancement of IGFBP-4 cleavage by PAPP-A and hypertrophic changes in cardiomyocytes accompanying CVD are interrelated, and PAPP-A appears to be one of the activators of the IGF-dependent processes in normal and hypertrophic-state cardiomyocytes.

CT-IGFBP-4 as a novel prognostic biomarker in acute heart failure.

AIMS: Insulin-like growth factor binding protein-4 (IGFBP-4) fragments have been shown to predict the risk of major adverse cardiovascular events, including segment-elevation myocardial infarction, in patients with acute coronary syndrome. We evaluated the prognostic value of the carboxy-terminal fragment of IGFBP-4 (CT-IGFBP-4) for all-cause mortality in emergency room patients with acute heart failure (AHF). METHODS AND RESULTS: CT-IGFBP-4, N-terminal pro brain natriuretic peptide (NT-proBNP), and C-reactive protein (CRP) were measured at admission from the lithium-heparin plasma of 156 patients with AHF. All-cause mortality was recorded for 1 year. Receiver operator characteristic (ROC) curves, Kaplan-Meier, and Cox proportional hazard ratio analyses were performed to evaluate the prognostic value of the various clinical variables, CT-IGFBP-4, NT-proBNP, CRP, and their combinations. During 1 year of follow-up, 52 (33.3%) patients died. CT-IGFBP-4 only weakly correlated with NT-proBNP (Pearson correlation coefficient r = 0.16, P = 0.044) and did not correlate with CRP (r = 0.08, P = 0.35), emphasizing the different nature of these biomarkers. The receiver operator characteristic area under the curve (ROC AUC) of CT-IGFBP-4 for the prediction of all-cause mortality (0.727) was significantly higher than that of NT-proBNP (0.680, P = 0.045) and CRP (0.669, P = 0.016). The combination of CT-IGFBP-4, NT-proBNP, and CRP predicted mortality significantly better (ROC AUC = 0.788) than any of the biomarkers alone (P < 0.01 for all). The addition of CT-IGFBP-4 to a clinical prediction model that included age, gender, systolic blood pressure, creatinine, and sodium levels, as well as the history of previous heart failure, coronary artery disease, and hypertension significantly improved the mortality risk prediction (ROC AUC 0.774 vs. 0.699, P = 0.025). Cox hazard analysis indicated that elevated CT-IGFBP-4 was independently associated with 1 year mortality (hazard ratio 3.26, P = 0.0008) after adjustment for age, gender, history of previous heart failure, coronary artery disease, hypertension, chronic kidney failure, history of diabetes, heart rate, haemoglobin, plasma sodium, NT-proBNP, CRP, cystatin C, and elevated cardiac troponin I or T. Patients with increased levels of either two or three of the biomarkers CT-IGFBP-4, NT-proBNP, and CRP had significantly higher mortality risk (adjusted hazard ratio 10.04, P < 0.0001) than patients with increased levels of one or none of the biomarkers. CONCLUSIONS: CT-IGFBP-4 was independently associated with all-cause mortality in patients with AHF. Compared with single biomarkers, the combination of CT-IGFBP-4, NT-proBNP, and CRP improved the prediction of all-cause mortality in patients with AHF.

Glycosylated and non-glycosylated NT-IGFBP-4 in circulation of acute coronary syndrome patients.

BACKGROUND: N-terminal and C-terminal proteolytic fragments of IGF binding protein 4 (NT-IGFBP-4 and CT-IGFBP-4) were recently shown to predict adverse cardiac events in acute coronary syndrome (ACS) patients. NT-IGFBP-4 and CT-IGFBP-4 are products of the pregnancy-associated plasma protein-A (PAPP-A)-mediated cleavage of IGFBP-4. It has been demonstrated that circulating IGFBP-4 is partially glycosylated in its N-terminal region, although the influence of this glycosylation on PAPP-A-mediated proteolysis and the ratio of glycosylated/non-glycosylated IGFBP-4 fragments in human blood remain unrevealed. The aims of this study were to investigate i) the presence of glycosylated NT-IGFBP-4 in the circulation, ii) the influence of the glycosylation of IGFBP-4 on its susceptibility to PAPP-A-mediated cleavage, and iii) the influence of glycosylation on NT-IGFBP-4 immunodetection. METHODS: Affinity purification was used for the extraction of IGFBP-4 and NT-IGFBP-4 from plasma samples. Purified proteins were quantified by Western blotting and specific sandwich immunoassays, while molecular masses were determined using mass spectrometry. RESULTS: Glycosylated NT-IGFBP-4 was identified in the blood of ACS patients. The fraction of glycosylated NT-IGFBP-4 in individual plasma samples was 9.8%-23.5% of the total levels of NT-IGFBP-4. PAPP-A-mediated proteolysis of glycosylated IGFBP-4 was 3-4 times less efficient (p < 0.001) than proteolysis of non-glycosylated protein. A sandwich fluoroimmunoassay that was designed for quantitative NT-IGFBP-4 measurements recognized both protein forms with the same efficiency. CONCLUSIONS: Although glycosylation suppresses PAPP-A-mediated IGFBP-4 cleavage, a considerable amount of glycosylated NT-IGFBP-4 is present in blood. Glycosylation does not influence NT-IGFBP-4 measurements using a specific sandwich immunoassay.

Characterization of endogenously circulating IGFBP-4 fragments-Novel biomarkers for cardiac risk assessment.

BACKGROUND: Recent findings show that circulating N- and C-terminal fragments of IGF-binding protein-4 (NT-IGFBP-4 and CT-IGFBP-4) can be utilized as biomarkers for cardiac risk assessment in acute coronary syndrome (ACS) patients. The fragments are thought to be the products of pregnancy-associated plasma protein A (PAPP-A)-dependent proteolysis. Two immunoassays for the measurement of IGFBP-4 fragments have been proposed. However, properties of the endogenous IGFBP-4 fragments that could influence the performance of the immunoassays were still not investigated. METHODS: NT- and CT-IGFBP-4 were extracted from pooled ACS plasma using affinity purification, and their concentrations were measured using sandwich immunoassays utilizing antibodies specific to their proteolytic neo-epitopes or internal epitopes. The extracted fragments were characterized by Western blots (WB) and mass-spectrometry. ACS plasma samples were analyzed by size exclusion chromatography (SEC). RESULTS: Immunoassays utilizing the neo-epitope-specific and the internal epitope-specific antibodies measured equal concentrations of the analyte in the endogenous IGFBP-4 fragments preparations. Only the 18 kDa NT-IGFBP-4 and 14 kDa CT-IGFBP-4 were detected in the WB analysis. Using mass-spectrometry, peaks corresponding to intact non-truncated and non-modified NT-IGFBP-4 (14626 Da) and CT-IGFBP-4 (11346 Da) were observed. The absence of complexed forms of IGFBP-4 in patients' plasma was demonstrated using SEC. CONCLUSIONS: Endogenous NT- and CT-IGFBP-4 from ACS patients' plasma correspond to the PAPP-A-derived IGFBP-4 fragments and do not undergo any truncation, modification, or complex formation in the patients' blood. Because of the demonstrated intact state of the circulating IGFBP-4 fragments, the neo-epitope-specific immunoassays perform reliably, allowing further clinical validation of these novel biomarkers.

Kinase-related protein/telokin inhibits Ca2+-independent contraction in Triton-skinned guinea pig taenia coli.

KRP (kinase-related protein), also known as telokin, has been proposed to inhibit smooth muscle contractility by inhibiting the phosphorylation of the rMLC (regulatory myosin light chain) by the Ca2+-activated MLCK (myosin light chain kinase). Using the phosphatase inhibitor microcystin, we show in the present study that KRP also inhibits Ca2+-independent rMLC phosphorylation and smooth muscle contraction mediated by novel Ca2+-independent rMLC kinases. Incubating KRP-depleted Triton-skinned taenia coli with microcystin at pCa>8 induced a slow contraction reaching 90% of maximal force (Fmax) at pCa 4.5 after approximately 25 min. Loading the fibres with KRP significantly slowed down the force development, i.e. the time to reach 50% of Fmax was increased from 8 min to 35 min. KRP similarly inhibited rMLC phosphorylation of HMM (heavy meromyosin) in vitro by MLCK or by the constitutively active MLCK fragment (61K-MLCK) lacking the myosin-docking KRP domain. A C-terminally truncated KRP defective in myosin binding inhibited neither force nor HMM phosphorylation. Phosphorylated KRP inhibited the rMLC phosphorylation of HMM in vitro and Ca2+-insensitive contractions in fibres similar to unphosphorylated KRP, whereby the phosphorylation state of KRP was not altered in the fibres. We conclude that (i) KRP inhibits not only MLCK-induced contractions, but also those elicited by Ca2+-independent rMLC kinases; (ii) phosphorylation of KRP does not modulate this effect; (iii) binding of KRP to myosin is essential for this inhibition; and (iv) KRP inhibition of rMLC phosphorylation is most probably due to the shielding of the phosphorylation site on the rMLC.

Processing of pro-B-type natriuretic peptide: furin and corin as candidate convertases.

BACKGROUND: B-type natriuretic peptide (BNP) and its N-terminal fragment (NT-proBNP) are the products of the enzyme-mediated cleavage of their precursor molecule, proBNP. The clinical significance of proBNP-derived peptides as biomarkers of heart failure has been explored thoroughly, whereas little is known about the mechanisms of proBNP processing. We investigated the role of 2 candidate convertases, furin and corin, in human proBNP processing. METHODS: We measured proBNP expression in HEK 293 and furin-deficient LoVo cells. We used a furin inhibitor and a furin-specific small interfering RNA (siRNA) to explore the implication of furin in proBNP processing. Recombinant proBNPs were incubated with HEK 293 cells transfected with the corin-expressing plasmid. We applied mass spectrometry to analyze the products of furin- and corin-mediated cleavage. RESULTS: Reduction of furin activity significantly impaired proBNP processing in HEK 293 cells. Furin-deficient LoVo cells were unable to process proBNP, whereas coexpression with furin resulted in effective proBNP processing. Mass spectrometric analysis revealed that the furin-mediated cleavage of proBNP resulted in BNP 1-32, whereas corin-mediated cleavage led to the production of BNP 4-32. Some portion of proBNP in the plasma of heart failure patients was not glycosylated in the cleavage site region and was susceptible to furin-mediated cleavage. CONCLUSIONS: Both furin and corin are involved in the proBNP processing pathway, giving rise to distinct BNP forms. The significance of the presence of unprocessed proBNP in circulation that could be cleaved by the endogenous convertases should be further investigated for better understanding BNP physiology.

Processing of pro-brain natriuretic peptide is suppressed by O-glycosylation in the region close to the cleavage site.

BACKGROUND: Processing of the brain natriuretic peptide (BNP) precursor, proBNP, is a convertase-dependent reaction that produces 2 molecules--the active BNP hormone and the N-terminal part of proBNP (NT-proBNP). Although proBNP was first described more than 15 years ago, very little is known about the cellular mechanism of its processing. The study of proBNP processing mechanisms is important, because processing impairments could be associated with the development of heart failure (HF). METHODS: The biochemical properties of recombinant proBNP and NT-proBNP and the same molecules derived from the blood of HF patients were analyzed by gel-filtration chromatography, site-directed mutagenesis, and different immunochemical methods with a panel of monoclonal antibodies (MAbs). RESULTS: Part of the proBNP molecule (amino acid residues 61-76) located near the cleavage site was inaccessible to specific MAbs because of the presence of O-glycans, whereas the same region in NT-proBNP was completely accessible. We demonstrated that a convertase (furin) could effectively cleave deglycosylated (but not intact) proBNP. Of several mutant proBNP forms produced in a HEK 293 cell line, only the T71A variant was effectively processed in the cell. CONCLUSIONS: Only proBNP that was not glycosylated in the region of the cleavage site could effectively be processed into BNP and NT-proBNP. Site-directed mutagenesis enabled us to ascertain the unique suppressing role of T71-bound O-glycan in proBNP processing.

Novel immunoassay for quantification of brain natriuretic peptide and its precursor in human blood.

BACKGROUND: Brain natriuretic peptide (BNP) is an unstable molecule that can rapidly lose immunologic activity in blood. Conventional sandwich BNP immunoassays use 2 antibodies specific to 2 different epitopes. Larger distances between epitopes are associated with a greater probability of proteolysis sites being located between the antibody-binding sites, and thus such assays have an increased susceptibility to underdetect BNP because of the increased likelihood of proteolytic degradation. The purpose of our study was to develop a sandwich immunoassay for the precise quantification of BNP and BNP precursor (proBNP) in human blood that is not susceptible to proteolysis. METHODS: Mice were immunized with an immune complex consisting of monoclonal antibody (MAb) 24C5 (specific for BNP peptide 11-22) and the entire BNP molecule. The MAb used in our assay (Ab-BNP2) recognizes the immune complex but neither free BNP nor MAb 24C5. RESULTS: We used MAbs 24C5 and Ab-BNP2 to develop a new type of sandwich BNP assay (the "single-epitope sandwich assay"), which requires only a short BNP fragment (fragment 11-22) for immunodetection. This assay recognizes both BNP and proBNP with the same efficiency and sensitivity and demonstrates both considerably less susceptibility to antigen degradation and greater stability of the measured antigen than conventional sandwich BNP immunoassays. CONCLUSIONS: We have developed this sensitive single-epitope sandwich assay for detecting BNP, proBNP, and their fragments in human blood. This assay appears promising for use in clinical studies to assist in triage, management, and outcomes assessment in heart failure patients.

Immunodetection of glycosylated NT-proBNP circulating in human blood.

BACKGROUND: Brain natriuretic peptide (BNP) or NT-proBNP (N-terminal fragment of BNP precursor) measurements are recommended as aids in diagnosis and prognosis of patients with heart failure. Recently it has been shown that proBNP is O-glycosylated in human blood. The goal of this study was to map sites on the NT-proBNP molecule that should be recognized by antibodies used in optimal NT-proBNP assays. METHODS: We analyzed endogenous NT-proBNP by several immunochemical methods using a broad panel of monoclonal antibodies specific to different epitopes of the NT-proBNP molecule. RESULTS: Treatment of endogenous NT-proBNP by a mixture of glycosidases resulted in significant improvement of the interaction between deglycosylated NT-proBNP and monoclonal antibodies (MAbs) specific to the mid-fragment of the molecule. MAbs specific to the N- and C-terminal parts of NT-proBNP (epitopes 13-24 and 63-76) were able to recognize glycosylated and deglycosylated protein with similar efficiency. CONCLUSIONS: The central part of endogenous NT-proBNP is glycosylated, making it almost "invisible" for the antibodies specific to the mid-fragment of the molecule. Thus sandwich assays using even one antibody (poly- or monoclonal) specific to the central part of the molecule could underestimate the real concentration of endogenous NT-proBNP. MAbs specific to the N- and C-terminal parts of NT-proBNP (epitopes 13-24 and 63-76) are the best candidates to be used in an assay for optimal NT-proBNP immunodetection.