Ivana Bulatovic

Local Control of Nuclear Calcium Signaling in Cardiac Myocytes by Perinuclear Microdomains of Sarcolemmal Insulin-Like Growth Factor 1 Receptors

Rationale: The ability of a cell to independently regulate nuclear and cytosolic Ca2+ signaling is currently attributed to the differential distribution of inositol 1,4,5-trisphosphate receptor channel isoforms in the nucleoplasmic versus the endoplasmic reticulum. In cardiac myocytes, T-tubules confer the necessary compartmentation of Ca2+ signals, which allows sarcomere contraction in response to plasma membrane depolarization, but whether there is a similar structure tunneling extracellular stimulation to control nuclear Ca2+ signals locally has not been explored. Objective: To study the role of perinuclear sarcolemma in selective nuclear Ca2+ signaling. Methods and Results: We report here that insulin-like growth factor 1 triggers a fast and independent nuclear Ca2+ signal in neonatal rat cardiac myocytes, human embryonic cardiac myocytes, and adult rat cardiac myocytes. This fast and localized response is achieved by activation of insulin-like growth factor 1 receptor signaling complexes present in perinuclear invaginations of the plasma membrane. The perinuclear insulin-like growth factor 1 receptor pool connects extracellular stimulation to local activation of nuclear Ca2+ signaling and transcriptional upregulation through the perinuclear hydrolysis of phosphatidylinositol 4,5-biphosphate inositol 1,4,5-trisphosphate production, nuclear Ca2+ release, and activation of the transcription factor myocyte-enhancing factor 2C. Genetically engineered Ca2+ buffers—parvalbumin—with cytosolic or nuclear localization demonstrated that the nuclear Ca2+ handling system is physically and functionally segregated from the cytosolic Ca2+ signaling machinery. Conclusions: These data reveal the existence of an inositol 1,4,5-trisphosphate–dependent nuclear Ca2+ toolkit located in direct apposition to the cell surface, which allows the local control of rapid and independent activation of nuclear Ca2+ signaling in response to an extracellular ligand.

Local Control of Nuclear Ca2+ Signalling in Cardiac Myocytes by Perinuclear Microdomains of Sarcolemmal IGF-1 Receptors

Rationale: The ability of a cell to independently regulate nuclear and cytosolic Ca2+ signaling is currently attributed to the differential distribution of inositol 1,4,5-trisphosphate receptor channel isoforms in the nucleoplasmic versus the endoplasmic reticulum. In cardiac myocytes, T-tubules confer the necessary compartmentation of Ca2+ signals, which allows sarcomere contraction in response to plasma membrane depolarization, but whether there is a similar structure tunneling extracellular stimulation to control nuclear Ca2+ signals locally has not been explored. Objective: To study the role of perinuclear sarcolemma in selective nuclear Ca2+ signaling. Methods and Results: We report here that insulin-like growth factor 1 triggers a fast and independent nuclear Ca2+ signal in neonatal rat cardiac myocytes, human embryonic cardiac myocytes, and adult rat cardiac myocytes. This fast and localized response is achieved by activation of insulin-like growth factor 1 receptor signaling complexes present in perinuclear invaginations of the plasma membrane. The perinuclear insulin-like growth factor 1 receptor pool connects extracellular stimulation to local activation of nuclear Ca2+ signaling and transcriptional upregulation through the perinuclear hydrolysis of phosphatidylinositol 4,5-biphosphate inositol 1,4,5-trisphosphate production, nuclear Ca2+ release, and activation of the transcription factor myocyte-enhancing factor 2C. Genetically engineered Ca2+ buffers—parvalbumin—with cytosolic or nuclear localization demonstrated that the nuclear Ca2+ handling system is physically and functionally segregated from the cytosolic Ca2+ signaling machinery. Conclusions: These data reveal the existence of an inositol 1,4,5-trisphosphate–dependent nuclear Ca2+ toolkit located in direct apposition to the cell surface, which allows the local control of rapid and independent activation of nuclear Ca2+ signaling in response to an extracellular ligand.

IgM phosphorylcholine antibodies inhibit cell death and constitute a strong protection marker for atherosclerosis development, particularly in combination with other auto-antibodies against modified LDL

BACKGROUND We have reported that anti-phosphorylcholine (anti-PC) IgM is a protection marker for human cardiovascular disease (CVD) and atherosclerosis. We here investigate the anti-PC autoantibodies in a well-defined cohort with regard to idiotype, atherosclerosis progression and mechanisms for its protective action. METHODS Serum levels and binding specificities of different anti-PC isotypes were determined in 226 hypertensive individuals enrolled in European Lacidipine Study on Atherosclerosis using ELISA. The mean of the maximum Intima-Media Thicknesses (IMT) in the far walls of common carotids and bifurcations was assessed at the time of inclusion, and four years afterwards. Apoptosis in immune cells was induced with lysophosphatidylcholine (LPC) and quantified using the MTT-assay. RESULTS Anti-PC IgM, IgA and IgG1 (but not IgG2) was negatively associated with IMT-progression. Combining anti-PC IgM with data on antibodies against oxidized- and malondialdehyde-modified LDL further strengthened this association. At very high levels, anti-PC IgM exhibited a striking negative association with atherosclerosis progression (OR 0.05; CI 0.006-0.40). Analysis of serum samples taken four years apart in study participants affirmed the stability of anti-PC IgM titers over time. Examination of fine specificities revealed that the protective isotypes (IgM, IgA and IgG1) are of the Group I idiotype whereas the non-protective IgG2 subclass was Group II. Anti-PC IgM inhibited LPC-induced cell death of immune cells. CONCLUSION Group I anti-PC antibodies, particularly of the IgM class, are independent protection markers for atherosclerosis progression. One potential mechanism of action is inhibition of LPC-induced cell cytotoxicity.

Ischemia-reperfusion injury and pregnancy initiate time-dependent and robust signs of up-regulation of cardiac progenitor cells.

To explore how cardiac regeneration and cell turnover adapts to disease, different forms of stress were studied for their effects on the cardiac progenitor cell markers c-Kit and Isl1, the early cardiomyocyte marker Nkx2.5, and mast cells. Adult female rats were examined during pregnancy, after myocardial infarction and ischemia-reperfusion injury with/out insulin like growth factor-1(IGF-1) and hepatocyte growth factor (HGF). Different cardiac sub-domains were analyzed at one and two weeks post-intervention, both at the mRNA and protein levels. While pregnancy and myocardial infarction up-regulated Nkx2.5 and c-Kit (adjusted for mast cell activation), ischemia-reperfusion injury induced the strongest up-regulation which occurred globally throughout the entire heart and not just around the site of injury. This response seems to be partly mediated by increased endogenous production of IGF-1 and HGF. Contrary to c-Kit, Isl1 was not up-regulated by pregnancy or myocardial infarction while ischemia-reperfusion injury induced not a global but a focal up-regulation in the outflow tract and also in the peri-ischemic region, correlating with the up-regulation of endogenous IGF-1. The addition of IGF-1 and HGF did boost the endogenous expression of IGF and HGF correlating to focal up-regulation of Isl1. c-Kit expression was not further influenced by the exogenous growth factors. This indicates that there is a spatial mismatch between on one hand c-Kit and Nkx2.5 expression and on the other hand Isl1 expression. In conclusion, ischemia-reperfusion injury was the strongest stimulus with both global and focal cardiomyocyte progenitor cell marker up-regulations, correlating to the endogenous up-regulation of the growth factors IGF-1 and HGF. Also pregnancy induced a general up-regulation of c-Kit and early Nkx2.5+ cardiomyocytes throughout the heart. Utilization of these pathways could provide new strategies for the treatment of cardiac disease.

Sublethal caspase activation promotes generation of cardiomyocytes from embryonic stem cells.

Generation of new cardiomyocytes is critical for cardiac repair following myocardial injury, but which kind of stimuli is most important for cardiomyocyte regeneration is still unclear. Here we explore if apoptotic stimuli, manifested through caspase activation, influences cardiac progenitor up-regulation and cardiomyocyte differentiation. Using mouse embryonic stem cells as a cellular model, we show that sublethal activation of caspases increases the yield of cardiomyocytes while concurrently promoting the proliferation and differentiation of c-Kit+/α-actininlow cardiac progenitor cells. A broad-spectrum caspase inhibitor blocked these effects. In addition, the caspase inhibitor reversed the mRNA expression of genes expressed in cardiomyocytes and their precursors. Our study demonstrates that sublethal caspase-activation has an important role in cardiomyocyte differentiation and may have significant implications for promoting cardiac regeneration after myocardial injury involving exogenous or endogenous cell sources.

Wnt/β-Catenin Stimulation and Laminins Support Cardiovascular Cell Progenitor Expansion from Human Fetal Cardiac Mesenchymal Stromal Cells

Summary The intrinsic regenerative capacity of human fetal cardiac mesenchymal stromal cells (MSCs) has not been fully characterized. Here we demonstrate that we can expand cells with characteristics of cardiovascular progenitor cells from the MSC population of human fetal hearts. Cells cultured on cardiac muscle laminin (LN)-based substrata in combination with stimulation of the canonical Wnt/β-catenin pathway showed increased gene expression of ISL1, OCT4, KDR, and NKX2.5. The majority of cells stained positive for PDGFR-α, ISL1, and NKX2.5, and subpopulations also expressed the progenitor markers TBX18, KDR, c-KIT, and SSEA-1. Upon culture of the cardiac MSCs in differentiation media and on relevant LNs, portions of the cells differentiated into spontaneously beating cardiomyocytes, and endothelial and smooth muscle-like cells. Our protocol for large-scale culture of human fetal cardiac MSCs enables future exploration of the regenerative functions of these cells in the context of myocardial injury in vitro and in vivo.

Human fetal cardiac progenitors: The role of stem cells and progenitors in the fetal and adult heart.

The human fetal heart is formed early during embryogenesis as a result of cell migrations, differentiation, and formative blood flow. It begins to beat around gestation day 22. Progenitor cells are derived from mesoderm (endocardium and myocardium), proepicardium (epicardium and coronary vessels), and neural crest (heart valves, outflow tract septation, and parasympathetic innervation). A variety of molecular disturbances in the factors regulating the specification and differentiation of these cells can cause congenital heart disease. This review explores the contribution of different cardiac progenitors to the embryonic heart development; the pathways and transcription factors guiding their expansion, migration, and functional differentiation; and the endogenous regenerative capacity of the adult heart including the plasticity of cardiomyocytes. Unfolding these mechanisms will become the basis for understanding the dynamics of specific congenital heart disease as well as a means to develop therapy for fetal as well as postnatal cardiac defects and heart failure.

Algorithm comparison for cardiac image fusion of coronary computed tomography angiography and 3D echocardiography

Treatment decision for coronary artery disease (CAD) is based on both morphological and functional information. Image fusion of coronary computed tomography angiography (CCTA) and three-dimensional echocardiography (3DE) could combine morphology and function into a single image to facilitate diagnosis. Three semi-automatic feature-based algorithms for CCTA/3DE registration were implemented and applied on CAD patients. Algorithms were verified and compared using landmarks manually identified by a cardiologist. All algorithms were found feasible for CCTA/3DE fusion.

Method comparison for cardiac image registration of coronary computed tomography angiography and 3-D echocardiography

Abstract. Treatment decision for coronary artery disease (CAD) is based on both morphological and functional information. Image fusion of coronary computed tomography angiography (CCTA) and three-dimensional echocardiography (3DE) could combine morphology and function into a single image to facilitate diagnosis. Three semiautomatic feature-based methods for CCTA/3DE registration were implemented and applied on CAD patients. Methods were verified and compared using landmarks manually identified by a cardiologist. All methods were found feasible for CCTA/3DE fusion.