Biomedical subjects

Marco Mongillo

Publications and source records attributed to Marco Mongillo.

10 recordsLinked to original sources

PubMed2006

Myocardial glucose transport and utilization in patients with type 2 diabetes mellitus, left ventricular dysfunction, and coronary artery disease.

OBJECTIVES: This research was designed to assess the effect of type 2 diabetes mellitus (T2DM) on myocardial glucose utilization in patients with heart failure secondary to coronary artery disease. BACKGROUND: Patients with T2DM and coronary artery disease have an increased morbidity and mortality compared with patients with coronary artery disease without diabetes that may relate to a reduction in the ability of the myocardium to utilize glucose. METHODS: Myocardial blood flow and glucose utilization were assessed during a hyperinsulinemic clamp by 18F-flurodeoxyglucose and positron emission tomography in 54 patients (19 with T2DM) with multivessel coronary artery disease and heart failure. In a subgroup of 18 patients, myocardial biopsies were obtained during coronary bypass surgery to assess glucose transporter (GLUT4) distribution and protein concentration, and compared with myocardium from transplant donor hearts. RESULTS: Myocardial blood flow was similar in patients without diabetes and those with T2DM. Myocardial glucose utilization was lower in patients with T2DM (0.34 +/- 0.16 vs. 0.47 +/- 0.24 micromol x min(-1) x g(-1), p = 0.0002) despite comparable plasma insulin concentrations and a higher blood glucose concentration. Extraction of glucose by the myocardium was reduced in patients with T2DM (7.1 +/- 3.1% vs. 13.5 +/- 5.2%, p < 0.01). Myocardial GLUT4 protein was similar in patients with and without T2DM (p = 0.75). CONCLUSIONS: Patients with coronary artery disease and heart failure exhibit myocardial insulin resistance, and this is greater in those with T2DM. This may limit the ability of the myocardium in patients with T2DM to withstand ischemia and may contribute to the increased cardiovascular morbidity and mortality in such patients.

Aged↗

PubMed2005

Compartmentalized phosphodiesterase-2 activity blunts beta-adrenergic cardiac inotropy via an NO/cGMP-dependent pathway.

beta-Adrenergic signaling via cAMP generation and PKA activation mediates the positive inotropic effect of catecholamines on heart cells. Given the large diversity of protein kinase A targets within cardiac cells, a precisely regulated and confined activity of such signaling pathway is essential for specificity of response. Phosphodiesterases (PDEs) are the only route for degrading cAMP and are thus poised to regulate intracellular cAMP gradients. Their spatial confinement to discrete compartments and functional coupling to individual receptors provides an efficient way to control local [cAMP]i in a stimulus-specific manner. By performing real-time imaging of cyclic nucleotides in living ventriculocytes we identify a prominent role of PDE2 in selectively shaping the cAMP response to catecholamines via a pathway involving beta3-adrenergic receptors, NO generation and cGMP production. In cardiac myocytes, PDE2, being tightly coupled to the pool of adenylyl cyclases activated by beta-adrenergic receptor stimulation, coordinates cGMP and cAMP signaling in a novel feedback control loop of the beta-adrenergic pathway. In this, activation of beta3-adrenergic receptors counteracts cAMP generation obtained via stimulation of beta1/beta2-adrenoceptors. Our study illustrates the key role of compartmentalized PDE2 in the control of catecholamine-generated cAMP and furthers our understanding of localized cAMP signaling.

Adenine↗

PubMed2005

Study of cyclic adenosine monophosphate microdomains in cells.

Cyclic adenosine monophosphate (cAMP) controls the physiological response to many diverse extracellular stimuli. To maintain signal specificity, cAMP-mediated signaling is finely tuned by means of a complex array of proteins that control the spatial and temporal dynamics of the second messenger within the cell. To unravel the way a cell encodes cAMP signals, new biosensors have recently been introduced that allow imaging of the second messenger in living cells with high spatial resolution. The more recent generation of such biosensors exploits the phenomenon of fluorescence resonance energy transfer between the green fluorescent protein- tagged subunits of a chimeric protein kinase A, as the way to visualize and measure the dynamic fluctuations of cAMP. This chapter describes the molecular basis on which such a genetically encoded cAMP sensor relies and the tools and methods required to perform cAMP measurements in living samples.

Animals↗

PubMed2004

cGMP catabolism by phosphodiesterase 5A regulates cardiac adrenergic stimulation by NOS3-dependent mechanism.

Beta-adrenergic agonists stimulate cardiac contractility and simultaneously blunt this response by coactivating NO synthase (NOS3) to enhance cGMP synthesis and activate protein kinase G (PKG-1). cGMP is also catabolically regulated by phosphodiesterase 5A (PDE5A). PDE5A inhibition by sildenafil (Viagra) increases cGMP and is used widely to treat erectile dysfunction; however, its role in the heart and its interaction with beta-adrenergic and NOS3/cGMP stimulation is largely unknown. In nontransgenic (control) murine in vivo hearts and isolated myocytes, PDE5A inhibition (sildenafil) minimally altered rest function. However, when the hearts or isolated myocytes were stimulated with isoproterenol, PDE5A inhibition was associated with a suppression of contractility that was coupled to elevated cGMP and increased PKG-1 activity. In contrast, NOS3-null hearts or controls with NOS inhibited by N(G)-nitro-L-arginine methyl ester, or soluble guanylate cyclase (sGC) inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one, showed no effect of PDE5A inhibition on beta-stimulated contractility or PKG-1 activation. This lack of response was not attributable to altered PDE5A gene or protein expression or in vitro PDE5A activity, but rather to an absence of sGC-generated cGMP specifically targeted to PDE5A catabolism and to a loss of PDE5A localization to z-bands. Re-expression of active NOS3 in NOS3-null hearts by adenoviral gene transfer restored PDE5A z-band localization and the antiadrenergic efficacy of PDE5A inhibition. These data support a novel regulatory role of PDE5A in hearts under adrenergic stimulation and highlight specific coupling of PDE5A catabolic regulation with NOS3-derived cGMP attributable to protein subcellular localization and targeted synthetic/catabolic coupling.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2004

In vivo monitoring of Ca(2+) uptake into mitochondria of mouse skeletal muscle during contraction.

Although the importance of mitochondria in patho-physiology has become increasingly evident, it remains unclear whether these organelles play a role in Ca(2+) handling by skeletal muscle. This undefined situation is mainly due to technical limitations in measuring Ca(2+) transients reliably during the contraction-relaxation cycle. Using two-photon microscopy and genetically expressed "cameleon" Ca(2+) sensors, we developed a robust system that enables the measurement of both cytoplasmic and mitochondrial Ca(2+) transients in vivo. We show here for the first time that, in vivo and under highly physiological conditions, mitochondria in mammalian skeletal muscle take up Ca(2+) during contraction induced by motor nerve stimulation and rapidly release it during relaxation. The mitochondrial Ca(2+) increase is delayed by a few milliseconds compared with the cytosolic Ca(2+) rise and occurs both during a single twitch and upon tetanic contraction.

Animals↗

PubMed2004

Fluorescence resonance energy transfer-based analysis of cAMP dynamics in live neonatal rat cardiac myocytes reveals distinct functions of compartmentalized phosphodiesterases.

Cardiac myocytes have provided a key paradigm for the concept of the compartmentalized cAMP generation sensed by AKAP-anchored PKA. Phosphodiesterases (PDEs) provide the sole route for degrading cAMP in cells and are thus poised to regulate intracellular cAMP gradients. PDE3 and PDE4 represent the major cAMP degrading activities in rat ventriculocytes. By performing real-time imaging of cAMP in situ, we establish the hierarchy of these PDEs in controlling cAMP levels in basal conditions and on stimulation with a beta-adrenergic receptor agonist. PDE4, rather than PDE3, appears to be responsible for modulating the amplitude and duration of the cAMP response to beta-agonists. PDE3 and PDE4 localize to distinct compartments and this may underpin their different functional roles. Our findings indicate the importance of distinctly localized PDE isoenzymes in determining compartmentalized cAMP signaling.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2004

Measuring dynamic changes in cAMP using fluorescence resonance energy transfer.

cAMP is a ubiquitous second messenger that controls numerous cellular events including movement, growth, metabolism, contraction, and synaptic plasticity. With the emerging concept of compartmentalization of cAMP-dependent signaling, a detailed study of the spatio-temporal intracellular dynamics of cAMP is required. Here we describe a new methodology for monitoring fluctuations of cAMP in living cells, based on the use of a genetically encoded biosensor. The regulatory and catalytic subunits of the main cAMP effector, the protein kinase A (PKA), fused with two suitable green fluorescent protein (GFP) mutants is used for measuring changes in fluorescence resonance energy transfer (FRET) that correlate with changes in intracellular cAMP levels. This method allows the study of cAMP fluctuations in living cells with high resolution both in time and in space.

Adenine Nucleotides↗

PubMed2003

Human MYO18B, a novel unconventional myosin heavy chain expressed in striated muscles moves into the myonuclei upon differentiation.

We have characterized a novel unconventional myosin heavy chain, named MYO18B, that appears to be expressed mainly in human cardiac and skeletal muscles and, at lower levels, in testis. MYO18B transcript is detected in all types of striated muscles but at much lower levels compared to class II sarcomeric myosins, and it is up regulated after in vitro differentiation of myoblasts into myotubes. Phylogenetic analysis shows that this myosin belongs to the recently identified class XVIII, however, unlike the other member of this class, it seems to be unique to Vertebrate since it contains two large amino acid domains of unknown function at the N and C-termini. Immunolocalization of MYO18B protein in skeletal muscle cells shows that this myosin heavy chain is located in the cytoplasm of undifferentiated myoblasts. After in vitro differentiation into myotubes, a fraction of this protein is accumulated in a subset of myonuclei. This nuclear localization was confirmed by immunofluorescence experiments on primary cardiomyocytes and adult muscle sections. In the cytoplasm MYO18B shows a punctate staining, both in cardiac and skeletal fibers. In some cases, cardiomyocytes show a partial sarcomeric pattern of MYO18B alternating that of alpha-actinin-2. In skeletal muscle the cytoplasmic MYO18B results much more evident in the fast type fibers.

Animals↗

PubMed2003

Looking forward to seeing calcium.

From molecules, single cells and tissues to whole organisms, our insights into Ca2+ signalling and the corresponding physiological phenomena are growing exponentially. Here, we describe the improvements that have been made in the development of the probes and instrumentation that are used for Ca2+ imaging and the expanding applications of Ca2+ imaging in basic and applied research.

Animals↗

PubMed2003

The Theodore Bücher lecture. Investigating signal transduction with genetically encoded fluorescent probes.

Ca2+ and cAMP are ubiquitous second messengers in eukaryotes and control numerous physiological responses ranging from fertilization to cell death induction. To distinguish between these different responses, their subtle regulation in time, space and amplitude is needed. Therefore, the characterization of the signalling process requires measurement of second messengers with tools of precise localization, high dynamic range and as little disturbance of cell physiology as possible. Recently, fluorescent proteins of marine jellyfish have given rise to a set of genetically encoded biosensors which fulfil these criteria and which have already led to important new insights into the subcellular handling of Ca2+ and cAMP. The use of these probes in combination with new microscopical methods such as two-photon microscopy now enables researchers to study second messenger signalling in intact tissues. In this review, the genetically encoded measurement probes and their origin are briefly introduced and some recent insights into the spatio-temporal complexity of both Ca2+ and cAMP signalling obtained with these tools are discussed.

Aequorin↗