ABSTRACT
In persons with dyslipidemia, a high residual risk of cardiovascular disease remains despite lipid lowering therapy. Current cardiovascular risk prediction mainly focuses on low-density lipoprotein cholesterol (LDL-c) levels, neglecting other contributing risk factors. Moreover, the efficacy of LDL-c lowering by statins resulting in reduced cardiovascular risk is only partially effective. Secondly, from a metrological viewpoint LDL-c falls short as a reliable measurand. Both direct and calculated LDL-c tests produce inaccurate test results at the low end under aggressive lipid lowering therapy. As LDL-c tests underperform both clinically and metrologically, there is an urging need for molecularly defined biomarkers. Over the years, apolipoproteins have emerged as promising biomarkers in the context of cardiovascular disease as they are the functional workhorses in lipid metabolism. Among these, apolipoprotein B (ApoB), present on all atherogenic lipoprotein particles, has demonstrated to clinically outperform LDL-c. Other apolipoproteins, such as Apo(a) - the characteristic apolipoprotein of the emerging risk factor lipoprotein(a) -, and ApoC-III - an inhibitor of triglyceride-rich lipoprotein clearance -, have attracted attention as well. To support personalized medicine, we need to move to molecularly defined risk markers, like the apolipoproteins. Molecularly defined diagnosis and molecularly targeted therapy require molecularly measured biomarkers. This review provides a summary of the scientific validity and (patho)physiological role of nine serum apolipoproteins, Apo(a), ApoB, ApoC-I, ApoC-II, ApoC-III, ApoE and its phenotypes, ApoA-I, ApoA-II, and ApoA-IV, in lipid metabolism, their association with cardiovascular disease, and their potential as cardiovascular risk markers when measured in a multiplex apolipoprotein panel.
ABSTRACT
BACKGROUND: Direct and calculated measures of lipoprotein fractions for cardiovascular risk assessment suffer from analytical inaccuracy in certain dyslipidemic and pathological states, most commonly hypertriglyceridemia. LC-MS/MS has proven suitable for multiplexed quantification and phenotyping of apolipoproteins. We developed and provisionally validated an automated assay for quantification of apolipoprotein (apo) A-I, B, C-I, C-II, C-III, and E and simultaneous qualitative assessment of apoE phenotypes. METHODS: We used 5 value-assigned human serum pools for external calibration. Serum proteins were denatured, reduced, and alkylated according to standard mass spectrometry-based proteomics procedures. After trypsin digestion, peptides were analyzed by LC-MS/MS. For each peptide, we measured 2 transitions. We compared LC-MS/MS results to those obtained by an immunoturbidimetric assay or ELISA. RESULTS: Intraassay CVs were 2.3%-5.5%, and total CVs were 2.5%-5.9%. The LC-MS/MS assay correlated (R = 0.975-0.995) with immunoturbidimetric assays with Conformité Européenne marking for apoA-I, apoB, apoC-II, apoC-III, and apoE in normotriglyceridemic (n = 54) and hypertriglyceridemic (n = 46) sera. Results were interchangeable for apoA-I ≤3.0 g/L (Deming slope 1.014) and for apoB-100 ≤1.8 g/L (Deming slope 1.016) and were traceable to higher-order standards. CONCLUSIONS: The multiplex format provides an opportunity for new diagnostic and pathophysiologic insights into types of dyslipidemia and allows a more personalized approach for diagnosis and treatment of lipid abnormalities.
Subject(s)
Apolipoproteins/blood , Automation , Dyslipidemias/blood , Dyslipidemias/diagnosis , Phenotype , Tandem Mass Spectrometry , Chromatography, High Pressure Liquid , Enzyme-Linked Immunosorbent Assay , Humans , Nephelometry and Turbidimetry , ProteomicsABSTRACT
Clinically actionable quantification of protein biomarkers by mass spectrometry (MS) requires analytical performance in concordance with quality specifications for diagnostic tests. Laboratory-developed tests should, therefore, be validated in accordance with EN ISO 15189:2012 guidelines for medical laboratories to demonstrate competence and traceability along the entire workflow, including the selected standardization strategy and the phases before, during, and after proteolysis. In this study, bias and imprecision of a previously developed MS method for quantification of serum apolipoproteins A-I (Apo A-I) and B (Apo B) were thoroughly validated according to Clinical and Laboratory Standards Institute (CLSI) guidelines EP15-A2 and EP09-A3, using 100 patient sera and either stable-isotope labeled (SIL) peptides or SIL-Apo A-I as internal standard. The systematic overview of error components assigned sample preparation before the first 4 h of proteolysis as major source (â¼85%) of within-sample imprecision without external calibration. No improvement in imprecision was observed with the use of SIL-Apo A-I instead of SIL-peptides. On the contrary, when the use of SIL-Apo A-I was combined with external calibration, imprecision improved significantly (from â¼9% to â¼6%) as a result of the normalization for matrix effects on linearity. A between-sample validation of bias in 100 patient sera further supported the presence of matrix effects on digestion completeness and additionally demonstrated specimen-specific biases associated with modified peptide sequences or alterations in protease activity. In conclusion, the presented overview of bias and imprecision components contributes to a better understanding of the sources of errors in MS-based protein quantification and provides valuable recommendations to assess and control analytical quality in concordance with the requirements for clinical use.
Subject(s)
Peptide Fragments/analysis , Peptide Fragments/standards , Proteomics/methods , Proteomics/standards , Blood Proteins/analysis , Blood Proteins/chemistry , Chromatography, Liquid , Humans , Isotope Labeling , Peptide Fragments/blood , Peptide Fragments/chemistry , Reproducibility of Results , Tandem Mass Spectrometry , TrypsinABSTRACT
Implementation of quantitative clinical chemistry proteomics (qCCP) requires targeted proteomics approaches, usually involving bottom-up multiple reaction monitoring-mass spectrometry (MRM-MS) with stable-isotope labeled standard (SIS) peptides, to move toward more accurate measurements. Two aspects of qCCP that deserve special attention are (1) proper calibration and (2) the assurance of consistent digestion. Here, we describe the evaluation of tryptic digestion efficiency by monitoring various signature peptides, missed cleavages, and modifications during proteolysis of apolipoprotein A-I and B in normo- and hypertriglyceridemic specimens. Absolute quantification of apolipoprotein A-I and B was performed by LC-MRM-MS with SIS peptide internal standards at two time points (4 and 20 h), using three native protein calibrators. Comparison with an immunoturbidimetric assay revealed recoveries of 99.4 ± 6.5% for apolipoprotein A-I and 102.6 ± 7.2% for apolipoprotein B after 4 h of trypsin digestion. Protein recoveries after 20 h trypsin incubation equaled 95.9 ± 6.9% and 106.0 ± 10.0% for apolipoproteins A-I and B, respectively. In conclusion, the use of metrologically traceable, native protein calibrators looks promising for accurate quantification of apolipoprotein A-I and B. Selection of rapidly formed peptides, that is, with no or minor missed cleavages, and the use of short trypsin incubation times for these efficiently cleaved peptides are likely to further reduce the variability introduced by trypsin digestion and to improve the traceability of test results to reach the desirable analytical performance for clinical chemistry application.
Subject(s)
Apolipoprotein A-I/blood , Apolipoproteins B/blood , Chromatography, Liquid/standards , Hypertriglyceridemia/blood , Mass Spectrometry/standards , Peptide Fragments/isolation & purification , Amino Acid Sequence , Apolipoprotein A-I/chemistry , Apolipoproteins B/chemistry , Calibration , Case-Control Studies , Humans , Molecular Sequence Data , Nephelometry and Turbidimetry , Peptide Fragments/chemistry , Proteolysis , Reference Standards , Reproducibility of Results , Trypsin/chemistryABSTRACT
Depolarization-induced automaticity (DIA) of cardiomyocytes is the property of those cells to generate pacemaker cell-like spontaneous electrical activity when subjected to a depolarizing current. This property provides a candidate mechanism for generation of pathogenic ectopy in cardiac tissue. The purpose of this study was to determine the biophysical mechanism of DIA in terms of the ion conductance properties of the cardiomyocyte membrane. First, we determined, by use of the conventional whole-cell patch-clamp technique, the membrane conductance and DIA properties of ventricular cardiomyocytes isolated from adult rat heart. Second, we reproduced and analysed DIA properties by using an adapted version of the experimentally based mathematical cardiomyocyte model of Pandit et al. (Biophys J 81:3029-3051 2001, Biophys J 84:832-841 2003) and Padmala and Demir (J Cardiovasc Electrophysiol 14:990-995 2003). DIA in 23 rat cardiomyocytes was a damped membrane potential oscillation with a variable number of action potentials and/or waves, depending on the strength of the depolarizing current and the particular cell. The adapted model was used to reconstruct the DIA properties of a particular cardiomyocyte from its whole-cell voltage-clamp currents. The main currents involved in DIA were an L-type calcium current (I CaL) and a slowly activating and inactivating Kv current (I ss), with linear (I B) and inward rectifier (I K1) currents acting as background currents and I Na and I t as modulators. Essential for DIA is a sufficiently large window current of a slowly inactivating I CaL combined with a critically sized repolarizing current I ss. Slow inactivation of I ss makes DIA transient. In conclusion, we established a membrane mechanism of DIA primarily based on I CaL, I ss and inward rectifier properties; this may be helpful in understanding cardiac ectopy and its treatment.
Subject(s)
Calcium Channels, L-Type/metabolism , Heart Ventricles/cytology , Ion Channel Gating , Membrane Potentials , Myocytes, Cardiac/cytology , Potassium Channels, Voltage-Gated/metabolism , Action Potentials , Animals , Electric Conductivity , Female , Models, Biological , Myocytes, Cardiac/metabolism , Rats , Rats, WistarSubject(s)
Antithrombin III/analysis , Chromatography, High Pressure Liquid , Tandem Mass Spectrometry , Amino Acid Sequence , Antithrombin III/metabolism , Antithrombin III/standards , Calibration , Chromatography, High Pressure Liquid/standards , Chymotrypsin/metabolism , Glycopeptides/analysis , Glycopeptides/isolation & purification , Glycopeptides/standards , Peptides/analysis , Peptides/isolation & purification , Peptides/standards , Reference Standards , Solid Phase Extraction , Tandem Mass Spectrometry/standards , Trypsin/metabolismABSTRACT
To improve patient outcome, point-of-care (POC) cardiac troponin I/T (cTn I/T) tests applied in a prehospital setting and/or emergency department might play a role as a substitute for central hospital laboratory high-sensitivity (hs) cTn I/T testing if their analytical and clinical performance are equivalent to central hospital laboratory hs cTn I/T tests and if they fulfill an unmet clinical need in the diagnostic work-up of patients with acute coronary syndrome (ACS). To date, current point-of-care (POC) cTn I/T tests are not yet sufficiently analytically sensitive and do not provide accurate and precise values in the reference range nor at the 99th percentile of a healthy reference population. Awaiting the development of improved hs POC cTn I/T tests, current POC cTn I/T tests should be combined with ECG as it takes several hours to detect a rise of cTn I/T in the circulation whereas ischemia-induced ECG changes may be observed soon after onset of chest pain. Although patients with acute ST-segment elevation myocardial infarction (STEMI) are generally diagnosed by ischemic symptoms and ECG only, hospitalized patients with non-STEMI and unstable angina pectoris (UAP) should preferentially be tested with ECG and central hospital laboratory hs cTn I/T tests unless the ECG has already demonstrated diagnostic changes. More evidence and future trials are needed to find out whether in patients with NSTE ACS hs cTn I/T tests should be combined with other tests, such as a test of B-type natriuretic peptide or NT-proBNP.
Subject(s)
Acute Coronary Syndrome/diagnosis , Acute Coronary Syndrome/epidemiology , Diagnosis, Computer-Assisted/methods , Electrocardiography/methods , Electrocardiography/statistics & numerical data , Evidence-Based Medicine , Troponin/blood , Acute Coronary Syndrome/blood , Biomarkers/blood , Humans , Prevalence , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
We previously showed that human cardiomyocyte progenitor cells (hCMPCs) injected after myocardial infarction (MI) had differentiated into cardiomyocytes in vivo 3 months after MI. Here, we investigated the short-term (2 weeks) effects of hCMPCs on the infarcted mouse myocardium. MI was induced in immunocompromised (NOD/scid) mice, immediately followed by intramyocardial injection of hCMPCs labelled with enhanced green fluorescent protein (hCMPC group) or vehicle only (control group). Sham-operated mice served as reference. Cardiac performance was measured 2 and 14 days after MI by magnetic resonance imaging at 9.4 T. Left ventricular (LV) pressure-volume measurements were performed at day 15 followed by extensive immunohistological analysis. Animals injected with hCMPCs demonstrated a higher LV ejection fraction, lower LV end-systolic volume and smaller relaxation time constant than control animals 14 days after MI. hCMPCs engrafted in the infarcted myocardium, did not differentiate into cardiomyocytes, but increased vascular density and proliferation rate in the infarcted and border zone area of the hCMPC group. Injected hCMPCs engraft into murine infarcted myocardium where they improve LV systolic function and attenuate the ventricular remodelling process 2 weeks after MI. Since no cardiac differentiation of hCMPCs was evident after 2 weeks, the observed beneficial effects were most likely mediated by paracrine factors, targeting amongst others vascular homeostasis. These results demonstrate that hCMPCs can be applied to repair infarcted myocardium without the need to undergo differentiation into cardiomyocytes.
Subject(s)
Cell Differentiation , Ischemia/therapy , Myoblasts/transplantation , Myocardial Infarction/therapy , Myocytes, Cardiac/cytology , Animals , Cells, Cultured , Green Fluorescent Proteins/metabolism , Humans , Ischemia/pathology , Magnetic Resonance Imaging , Male , Mice , Mice, Inbred NOD , Mice, SCID , Myoblasts/cytology , Myocardial Infarction/pathology , Ventricular Function, Left , Ventricular RemodelingABSTRACT
Phosphodiesterase (PDE) 4 inhibitors are potent anti-inflammatory drugs with antihypertensive properties, and their therapeutic role in bronchopulmonary dysplasia (BPD) is still controversial. We studied the role of PDE4 inhibition with piclamilast on normal lung development and its therapeutic value on pulmonary hypertension (PH) and right ventricular hypertrophy (RVH) in neonatal rats with hyperoxia-induced lung injury, a valuable model for premature infants with severe BPD. The cardiopulmonary effects of piclamilast treatment (5 mg·kg(-1)·day(-1)) were investigated in two models of experimental BPD: 1) daily treatment during continuous exposure to hyperoxia for 10 days; and 2) late treatment and injury-recovery in which pups were exposed to hyperoxia or room air for 9 days, followed by 9 or 42 days of recovery in room air combined with treatment started on day 6 of oxygen exposure until day 18. Prophylactic piclamilast treatment reduced pulmonary fibrin deposition, septum thickness, arteriolar wall thickness, arteriolar vascular smooth muscle cell proliferation and RVH, and prolonged survival. In the late treatment and injury-recovery model, hyperoxia caused persistent aberrant alveolar and vascular development, PH, and RVH. Treatment with piclamilast in both models reduced arteriolar wall thickness, attenuated RVH, and improved right ventricular function in the injury recovery model, but did not restore alveolarization or angiogenesis. Treatment with piclamilast did not show adverse cardiopulmonary effects in room air controls in both models. In conclusion, PDE4 inhibition attenuated and partially reversed PH and RVH, but did not advance alveolar development in neonatal rats with hyperoxic lung injury or affect normal lung and heart development.
Subject(s)
Benzamides , Bronchopulmonary Dysplasia , Heart Injuries/drug therapy , Hyperoxia/drug therapy , Phosphodiesterase 4 Inhibitors , Pyridines , Animals , Animals, Newborn , Benzamides/pharmacology , Benzamides/therapeutic use , Bronchopulmonary Dysplasia/drug therapy , Bronchopulmonary Dysplasia/pathology , Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Disease Models, Animal , Heart/drug effects , Heart/physiopathology , Heart Injuries/physiopathology , Humans , Hyperoxia/physiopathology , Hypertension, Pulmonary/drug therapy , Hypertension, Pulmonary/physiopathology , Hypertrophy, Right Ventricular/drug therapy , Hypertrophy, Right Ventricular/physiopathology , Infant, Newborn , Lung/drug effects , Lung/physiopathology , Oxidative Stress/drug effects , Oxygen/pharmacology , Phosphodiesterase 4 Inhibitors/pharmacology , Phosphodiesterase 4 Inhibitors/therapeutic use , Pulmonary Alveoli/drug effects , Pulmonary Alveoli/growth & development , Pyridines/pharmacology , Pyridines/therapeutic use , RatsABSTRACT
RATIONALE: Pulmonary hypertension (PH) is characterized by progressive increase in pulmonary artery pressure leading to right ventricular (RV) hypertrophy, RV failure, and death. Current treatments only temporarily reduce severity of the disease, and an ideal therapy is still lacking. OBJECTIVES: Estrogen pretreatment has been shown to attenuate development of PH. Because PH is not often diagnosed early, we examined if estrogen can rescue preexisting advanced PH. METHODS: PH was induced in male rats with monocrotaline (60 mg/kg). At Day 21, rats were either treated with 17-ß estradiol or estrogen (E2, 42.5 µg/kg/d), estrogen receptor-ß agonist (diarylpropionitrile, 850 µg/kg/d), or estrogen receptor α-agonist (4,4',4"-[4-Propyl-(1H)-pyrazole-1,3,5-triyl] trisphenol, 850 µg/kg/d) for 10 days or left untreated to develop RV failure. Serial echocardiography, cardiac catheterization, immunohistochemistry, Western blot, and real-time polymerase chain reaction were performed. MEASUREMENTS AND MAIN RESULTS: Estrogen therapy prevented progression of PH to RV failure and restored lung and RV structure and function. This restoration was maintained even after removal of estrogen at Day 30, resulting in 100% survival at Day 42. Estradiol treatment restored the loss of blood vessels in the lungs and RV. In the presence of angiogenesis inhibitor TNP-470 (30 mg/kg) or estrogen receptor-ß antagonist (PHTPP, 850 µg/kg/d), estrogen failed to rescue PH. Estrogen receptor-ß selective agonist was as effective as estrogen in rescuing PH. CONCLUSIONS: Estrogen rescues preexisting severe PH in rats by restoring lung and RV structure and function that are maintained even after removal of estrogen. Estrogen-induced rescue of PH is associated with stimulation of cardiopulmonary neoangiogenesis, suppression of inflammation, fibrosis, and RV hypertrophy. Furthermore, estrogen rescue is likely mediated through estrogen receptor-ß.
Subject(s)
Estrogens/therapeutic use , Hypertension, Pulmonary/drug therapy , Animals , Blood Pressure/drug effects , Blotting, Western , Estradiol/administration & dosage , Heart/drug effects , Male , Pulmonary Artery/drug effects , Rats , Rats, Sprague-Dawley , Real-Time Polymerase Chain Reaction , Severity of Illness IndexABSTRACT
BACKGROUND: the presence of atrial fibrillation (AF) is related to increased levels of natriuretic peptides. In addition, increased natriuretic peptide levels are predictive of the development of AF. However, the role of natriuretic peptides to predict recurrence of AF after radiofrequency catheter ablation (RFCA) is controversial. OBJECTIVE: the study aimed to investigate the role of natriuretic peptides in the prediction of AF recurrence after RFCA for AF. METHODS: pre-procedural amino-terminal pro-atrial natriuretic peptide (NT-proANP) and amino-terminal-pro-B-type natriuretic peptide (NT-proBNP) plasma levels were determined in 87 patients undergoing RFCA for symptomatic drug-refractory AF. In addition, a comprehensive clinical and echocardiographic evaluation was performed at baseline. Left atrial volumes, left ventricular volumes, and function (systolic and diastolic) were assessed. During a 6-month follow-up period, AF recurrence was monitored and defined as any registration of AF on electrocardiogram or an episode of AF longer than 30 seconds on 24-hour Holter monitoring. The role of natriuretic peptide plasma levels to predict AF recurrence after RFCA was studied. RESULTS: During follow-up, 66 patients (76%) maintained sinus rhythm, whereas 21 patients (24%) had AF recurrence. Patients with AF recurrence had higher baseline natriuretic peptide levels than patients who maintained sinus rhythm (NT-proANP 3.19 nmol/L [2.55-4.28] vs 2.52 nmol/L [1.69-3.55], P = .030; NT-proBNP 156.4 pg/mL [64.1-345.3] vs 84.6 pg/mL [43.3-142.7], P = .036). However, NT-proBNP was an independent predictor of AF recurrence, whereas NT-proANP was not. Moreover, NT-proBNP had an incremental value over echocardiographic characteristics to predict AF recurrence after RFCA. CONCLUSION: baseline NT-proBNP plasma level is an independent predictor of AF recurrence after RFCA.
Subject(s)
Atrial Fibrillation/blood , Catheter Ablation , Natriuretic Peptide, Brain/blood , Peptide Fragments/blood , Aged , Atrial Fibrillation/physiopathology , Atrial Fibrillation/surgery , Biomarkers/blood , Electrocardiography, Ambulatory , Enzyme-Linked Immunosorbent Assay , Female , Follow-Up Studies , Humans , Male , Middle Aged , Postoperative Period , Prognosis , Protein Precursors , Recurrence , Time FactorsABSTRACT
INTRODUCTION: Guidelines of management of dyslipidemias and prevention of cardiovascular disease (CVD) are based on firm scientific evidence obtained by randomized controlled trials (RCTs). However, the role of elevated low-density lipoprotein-cholesterol (LDL-C)as a risk factor of CVD and therapies to lower LDL-C are frequently disputed by colleagues who disagree with the conclusions of the RCTs published. This review focuses on this dispute, and evaluates the current approach of management of dyslipidemias and CVD prevention to find modern alternatives for more precise diagnosis and therapy of dyslipidemic patients. AREAS COVERED: Recent interest in lipoprotein(a) (Lp(a)) and remnants lipoproteins and in therapies that do not influence LDL-C levels primarily, such as anti-inflammatory drugs and icosapent ethyl, has revitalized our concern to optimize the care for patients with increased CVD risk without focusing simply on reduction of LDL-C by therapy with statins, ezitemibe, and proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitors. EXPERT OPINION: The limited characterization of study populations by measurement of total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C) and triglycerides (TG) followed by measurement or calculation of LDL-C should be extended by a more integral approach in order to realize precision diagnostics and precision medicine, for the sake of personalized patient care.
Subject(s)
Anticholesteremic Agents , Cardiovascular Diseases , Dyslipidemias , Hydroxymethylglutaryl-CoA Reductase Inhibitors , Cardiovascular Diseases/etiology , Cardiovascular Diseases/prevention & control , Dyslipidemias/complications , Dyslipidemias/drug therapy , Heart Disease Risk Factors , Humans , Lipoprotein(a) , PCSK9 Inhibitors , Precision Medicine , Proprotein Convertase 9ABSTRACT
Previously, our research group showed that integrin stimulation induces release of cardiac troponin I from viable neonatal rat ventricular cardiomyocytes (NRCMs), but would it also stimulate uptake of exogenous macromolecules? For this purpose, beating NRCMs were incubated without or with an RGD motif-containing peptide (GRGDS) to stimulate integrins in the presence of Texas Red-conjugated ovalbumin (OTR; 45 kDa) or dextran (DTR; 70 kDa). After incubation periods of 8, 16 and 24 hours endocytosis of red label was quantified by fluorescence microscopy. Uptake of OTR and DTR by NRCMs was intensified by GRGDS treatment (p for trend <0.001 and 0.019, respectively) and increased with duration of incubation (p<0.001 for both). The GRGDS-induced uptake of OTR by NRCMs correlated positively with OTR concentration (p<0.001). Experiments with pharmacological inhibitors of endocytosis indicated that in the absence of GRGDS, NRCMs take up OTR by the clathrin-mediated pathway of endocytosis while the GRGDS-dependent OTR uptake occurs by macropinocytosis. Cultures of NRCMs that were stretched cyclically showed â4-fold increased uptake of OTR compared to stationary NRCM cultures. Immunofluorescence microscopy revealed that the dysferlin-positive plasma membrane (PM) areas in beating GRGDS-treated NRCMs were â3-fold larger than in contracting NRCMs incubated with vehicle (p<0.001). However, in non-beating NRCMs exposure to GRGDS did not induce larger dysferlin-positive PM areas, nor did it stimulate uptake of OTR. After inhibition of dysferlin expression by short hairpin RNA-mediated RNA interference, OTR uptake by contracting NRCMs could no longer be stimulated via GRGDS treatment. We conclude that in NRCMs, stimulation of integrins by RGD motif-containing peptides or stretch cause uptake of labeled macromolecules. The latter process appears to depend on the contractile behavior of the NRCMs and on the PM repair protein dysferlin, probably because of its role in macropinocytosis.
Subject(s)
Integrins/metabolism , Myocytes, Cardiac/metabolism , Animals , Dextrans/chemistry , Dextrans/metabolism , Endocytosis , Fluorescent Dyes/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Membrane Proteins/physiology , Microscopy, Fluorescence , Muscle Proteins/genetics , Muscle Proteins/metabolism , Muscle Proteins/physiology , Myocytes, Cardiac/physiology , Oligopeptides/pharmacology , Ovalbumin/chemistry , Ovalbumin/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Rats , Rats, Wistar , Xanthenes/chemistryABSTRACT
Nitric oxide (NO) produced in the heart by nitric oxide synthase (NOS) is a highly reactive signaling molecule and an important modulator of myocardial function. NOS catalyzes the conversion of L: -arginine to L: -citrulline and NO but under particular circumstances reactive oxygen species (ROS) can be formed instead of NO (uncoupling). In the heart, three NOS isoforms are present: neuronal NOS (nNOS, NOS1) and endothelial NOS (eNOS, NOS3) are constitutively present enzymes in distinct subcellular locations within cardiomyocytes, whereas inducible NOS (iNOS, NOS2) is absent in the healthy heart, but its expression is induced by pro-inflammatory mediators. In the tissue, NO has two main effects: (i) NO stimulates the activity of guanylate cyclase, leading to cGMP generation and activation of protein kinase G, and (ii) NO nitrosylates tyrosine and thiol-groups of cysteine in proteins. Upon nitrosylation, proteins may change their properties. Changes in (i) NOS expression and activity, (ii) subcellular compartmentation of NOS activity, and (iii) the occurrence of uncoupling may lead to multiple NO-induced effects, some of which being particularly evident during myocardial overload as occurs during aortic constriction and myocardial infarction. Many of these NO-induced effects are considered to be cardioprotective but particularly if NOS becomes uncoupled, formation of ROS in combination with a low NO bioavailability predisposes for cardiac damage.
Subject(s)
Nitric Oxide Synthase/physiology , Nitric Oxide/metabolism , Cardiomegaly/enzymology , Heart Failure/enzymology , Myocardium/enzymology , Nitric Oxide Synthase Type II/physiology , Nitric Oxide Synthase Type III/physiologyABSTRACT
Alignment of cardiomyocytes (CMCs) contributes to the anisotropic (direction-related) tissue structure of the heart, thereby facilitating efficient electrical and mechanical activation of the ventricles. This study aimed to investigate the effects of forced alignment of stem cells during cardiomyogenic differentiation on their functional integration with CMC cultures. Labeled neonatal rat (nr) mesenchymal stem cells (nrMSCs) were allowed to differentiate into functional heart muscle cells in different cell-alignment patterns during 10 days of coculture with nrCMCs. Development of functional cellular properties was assessed by measuring impulse transmission across these stem cells between 2 adjacent nrCMC fields, cultured onto microelectrode arrays and previously separated by a laser-dissected channel (230+/-10 microm) for nrMSC transplantation. Coatings in these channels were microabraded in a direction (1) parallel or (2) perpendicular to the channel or were (3) left unabraded to establish different cell patterns. Application of cells onto microabraded coatings resulted in anisotropic cell alignment within the channel. Application on unabraded coatings resulted in isotropic (random) alignment. After coculture, conduction across seeded nrMSCs occurred from day 1 (perpendicular and isotropic) or day 6 (parallel) onward. Conduction velocity across nrMSCs at day 10 was highest in the perpendicular (11+/-0.9 cm/sec; n=12), intermediate in the isotropic (7.1+/-1 cm/sec; n=11) and lowest in the parallel configuration (4.9+/-1 cm/sec; n=11) (P<0.01). nrCMCs and fibroblasts served as positive and negative control, respectively. Also, immunocytochemical analysis showed alignment-dependent increases in connexin 43 expression. In conclusion, forced alignment of nrMSCs undergoing cardiomyogenic differentiation affects the time course and degree of functional integration with surrounding cardiac tissue.
Subject(s)
Cell Communication/physiology , Cell Differentiation/physiology , Heart Conduction System/physiology , Mesenchymal Stem Cells/cytology , Myocytes, Cardiac/cytology , Animals , Cells, Cultured , Coculture Techniques , Connexin 43/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Male , Mesenchymal Stem Cells/metabolism , Microelectrodes , Models, Animal , Myocardium/metabolism , Myocytes, Cardiac/metabolism , Patch-Clamp Techniques , Rats , Rats, WistarABSTRACT
BACKGROUND: The developmental mechanisms underlying the persistence of myocardial accessory atrioventricular pathways (APs) that bypass the annulus fibrosis are mainly unknown. In the present study, we investigated the role of epicardium-derived cells (EPDCs) in annulus fibrosis formation and the occurrence of APs. METHODS AND RESULTS: EPDC migration was mechanically inhibited by in ovo microsurgery in quail embryos. In ovo ECGs were recorded in wild-type (n=12) and EPDC-inhibited (n=12) hearts at Hamburger-Hamilton (HH) stages 38 to 42. Subsequently, in these EPDC-inhibited hearts (n=12) and in additional wild-type hearts (n=45; HH 38-42), ex ovo extracellular electrograms were recorded. Electrophysiological data were correlated with differentiation markers for cardiomyocytes (MLC2a) and fibroblasts (periostin). In ovo ECGs showed significantly shorter PR intervals in EPDC-inhibited hearts (45+/-10 ms) than in wild-type hearts (55+/-8 ms, 95% CI 50 to 60 ms, P=0.030), whereas the QRS durations were significantly longer in EPDC-inhibited hearts (29+/-14 versus 19+/-2 ms, 95% CI 18 to 21 ms, P=0.011). Furthermore, ex ovo extracellular electrograms (HH 38-42) displayed base-first ventricular activation in 44% (20/45) of wild-type hearts, whereas in all EPDC-inhibited hearts (100%, 12/12), the ventricular base was activated first (P<0.001). Small periostin- and MLC2a-positive APs were found mainly in the posteroseptal region of both wild-type and EPDC-inhibited hearts. Interestingly, in all (n=10) EPDC-inhibited hearts, additional large periostin-negative and MLC2a-positive APs were found in the right and left lateral free wall coursing through marked isolation defects in the annulus fibrosis until the last stages of embryonic development. CONCLUSIONS: EPDCs play an important role in annulus fibrosis formation. EPDC outgrowth inhibition may result in marked defects in the fibrous annulus with persistence of large APs, which results in ventricular preexcitation on ECG. These APs may provide a substrate for postnatally persistent reentrant arrhythmias.
Subject(s)
Bundle of His , Fibrosis/pathology , Pericardium/cytology , Animals , Cell Movement , Electrocardiography , Embryo, Nonmammalian , Fibroblasts , Fibrosis/etiology , Myocytes, Cardiac , Pericardium/embryology , Pre-Excitation Syndromes/etiology , Pre-Excitation Syndromes/pathology , QuailABSTRACT
Pulmonary arterial hypertension (PAH) is a chronic lung disease that leads to right ventricular (RV) hypertrophy (RVH), remodeling, and failure. We tested treatment with bone marrow-derived mesenchymal stem cells (MSCs) obtained from donor rats with monocrotaline (MCT)-induced PAH to recipient rats with MCT-induced PAH on pulmonary artery pressure, lung pathology, and RV function. This model was chosen to mimic autologous MSC therapy. On day 1, PAH was induced by MCT (60 mg/kg) in 20 female Wistar rats. On day 14, rats were treated with 10(6) MSCs intravenously (MCT + MSC) or with saline (MCT60). MSCs were obtained from donor rats with PAH at 28 days after MCT. A control group received saline on days 1 and 14. On day 28, the RV function of recipient rats was assessed, followed by isolation of the lungs and heart. RVH was quantified by the weight ratio of the RV/(left ventricle + interventricular septum). MCT induced an increase of RV peak pressure (from 27 + or - 5 to 42 +/- 17 mmHg) and RVH (from 0.25 + or - 0.04 to 0.47 + or - 0.12), depressed the RV ejection fraction (from 56 + or - 11 to 43 + or - 6%), and increased lung weight (from 0.96 + or - 0.15 to 1.66 + or - 0.32 g), including thickening of the arteriolar walls and alveolar septa. MSC treatment attenuated PAH (31 + or - 4 mmHg) and RVH (0.32 + or - 0.07), normalized the RV ejection fraction (52 + or - 5%), reduced lung weight (1.16 + or - 0.24 g), and inhibited the thickening of the arterioles and alveolar septa. We conclude that the application of MSCs from donor rats with PAH reduces RV pressure overload, RV dysfunction, and lung pathology in recipient rats with PAH. These results suggest that autologous MSC therapy may alleviate cardiac and pulmonary symptoms in PAH patients.
Subject(s)
Hypertension, Pulmonary/surgery , Hypertrophy, Right Ventricular/prevention & control , Lung/pathology , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Myocardium/pathology , Ventricular Dysfunction, Right/prevention & control , Ventricular Function, Right , Animals , Arterioles/pathology , Cardiac Output , Cell Differentiation , Cell Proliferation , Cells, Cultured , Disease Models, Animal , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Female , Heart Rate , Hypertension, Pulmonary/chemically induced , Hypertension, Pulmonary/pathology , Hypertension, Pulmonary/physiopathology , Hypertrophy, Right Ventricular/chemically induced , Hypertrophy, Right Ventricular/pathology , Hypertrophy, Right Ventricular/physiopathology , Inflammation Mediators/metabolism , Lung/blood supply , Lung/metabolism , Lung/physiopathology , Mesenchymal Stem Cells/metabolism , Monocrotaline , Myocardial Contraction , Myocardium/metabolism , Pulmonary Alveoli/pathology , Pulmonary Artery/pathology , RNA, Messenger/metabolism , Rats , Rats, Wistar , Recovery of Function , Stroke Volume , Time Factors , Transplantation, Homologous , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Ventricular Dysfunction, Right/chemically induced , Ventricular Dysfunction, Right/pathology , Ventricular Dysfunction, Right/physiopathology , Ventricular Pressure , Ventricular RemodelingABSTRACT
BACKGROUND: We investigated whether (1) monocrotaline(MCT)-induced right ventricular (RV) dilatation is associated with re-expression of myocardial tenascin-C (TNC), (2) elevated plasma TNC levels can be used as a marker of ventricular dilatation, and (3) MCT-induced RV dilatation is associated with alterations of other remodeling-related proteins. METHODS: Rats were treated with MCT in low dose (30 mg/kg, MCT30, n=10) to induce compensated RV hypertrophy, in high dose (80 mg/kg, MCT80, n=11) to induce RV failure, and with saline as control (CONT, n=9). After 4 weeks, RV function was assessed. TNC gene expression, protein levels, and plasma levels were assayed. Myocardial gene expression of integrin subunit alpha6 and beta1, and myocardial proMMP2 and proMMP9 levels were assessed. RESULTS: TNC gene expression in the RV of MCT80 rats was significantly upregulated compared with RV of CONT (4-fold, p<0.001) and MCT30 rats (3-fold, p<0.01), whereas TNC gene expression was very low in LV and IVS of MCT80 rats, and absent in those of CONT and MCT30 rats. Myocardial TNC protein levels were only observed in MCT80 rats, and were higher in RV (0.62+/-0.64 ng/mg) than in LV (0.21+/-0.44 ng/mg) or IVS (0.21+/-0.09 ng/mg) (p<0.01, RV vs LV and IVS). Plasma levels of TNC were significantly elevated in MCT80 rats compared with CONT (p<0.05). RV volumes correlated positively with TNC plasma levels and RV ejection fraction correlated negatively with TNC plasma levels. MCT-induced RV failure was also associated with a significant downregulation of integrin alpha6 gene expression (by 48%, p<0.01). CONCLUSIONS: MCT-induced RV failure is associated with an upregulation of TNC gene expression, resulting in re-expression of myocardial TNC protein levels, and elevated TNC plasma levels. As RV ejection fraction correlated significantly with TNC plasma levels, TNC plasma levels may serve as a marker of ventricular failure. De-adhesive properties of TNC in combination with a downregulation of integrin alpha6 may have caused cardiomyocyte slippage, leading to adverse ventricular remodeling.
Subject(s)
Heart Ventricles/physiopathology , Hypertrophy, Right Ventricular/physiopathology , Myocardium/metabolism , Tenascin/metabolism , Ventricular Dysfunction, Right/physiopathology , Animals , Blood Pressure , Gene Expression , Heart Failure/metabolism , Hypertrophy, Right Ventricular/chemically induced , Hypertrophy, Right Ventricular/pathology , Immunohistochemistry , Integrins/metabolism , Male , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Monocrotaline , Rats , Rats, Wistar , Tenascin/blood , Time Factors , Ventricular Dysfunction, Right/chemically induced , Ventricular Function, Right/physiology , Ventricular Pressure , Ventricular Remodeling/physiologyABSTRACT
BACKGROUND: Phosphodiesterase-5 inhibition with sildenafil has been used to treat severe pulmonary hypertension and bronchopulmonary dysplasia (BPD), a chronic lung disease in very preterm infants who were mechanically ventilated for respiratory distress syndrome. METHODS: Sildenafil treatment was investigated in 2 models of experimental BPD: a lethal neonatal model, in which rat pups were continuously exposed to hyperoxia and treated daily with sildenafil (50-150 mg/kg body weight/day; injected subcutaneously) and a neonatal lung injury-recovery model in which rat pups were exposed to hyperoxia for 9 days, followed by 9 days of recovery in room air and started sildenafil treatment on day 6 of hyperoxia exposure. Parameters investigated include survival, histopathology, fibrin deposition, alveolar vascular leakage, right ventricular hypertrophy, and differential mRNA expression in lung and heart tissue. RESULTS: Prophylactic treatment with an optimal dose of sildenafil (2 x 50 mg/kg/day) significantly increased lung cGMP levels, prolonged median survival, reduced fibrin deposition, total protein content in bronchoalveolar lavage fluid, inflammation and septum thickness. Treatment with sildenafil partially corrected the differential mRNA expression of amphiregulin, plasminogen activator inhibitor-1, fibroblast growth factor receptor-4 and vascular endothelial growth factor receptor-2 in the lung and of brain and c-type natriuretic peptides and the natriuretic peptide receptors NPR-A, -B, and -C in the right ventricle. In the lethal and injury-recovery model we demonstrated improved alveolarization and angiogenesis by attenuating mean linear intercept and arteriolar wall thickness and increasing pulmonary blood vessel density, and right ventricular hypertrophy (RVH). CONCLUSION: Sildenafil treatment, started simultaneously with exposure to hyperoxia after birth, prolongs survival, increases pulmonary cGMP levels, reduces the pulmonary inflammatory response, fibrin deposition and RVH, and stimulates alveolarization. Initiation of sildenafil treatment after hyperoxic lung injury and continued during room air recovery improves alveolarization and restores pulmonary angiogenesis and RVH in experimental BPD.