[Show abstract][Hide abstract] ABSTRACT: We recently developed a method to measure mitochondrial proteome dynamics with heavy water ((2)H2O)-based metabolic labeling and high resolution mass spectrometry. We reported the half-lives and synthesis rates of several proteins in the two cardiac mitochondrial subpopulations, subsarcolemmal and interfibrillar (SSM and IFM), in Sprague Dawley rats. In the present study, we tested the hypothesis that the mitochondrial protein synthesis rate is reduced in heart failure, with possible differential changes in SSM versus IFM. Six to seven week old male Sprague Dawley rats underwent transverse aortic constriction (TAC) and developed moderate heart failure after 22weeks. Heart failure and sham rats of the same age received heavy water (5% in drinking water) for up to 80days. Cardiac SSM and IFM were isolated from both groups and the proteins were separated by 1D gel electrophoresis. Heart failure reduced protein content and increased the turnover rate of several proteins involved in fatty acid oxidation, electron transport chain and ATP synthesis, while it decreased the turnover of other proteins, including pyruvate dehydrogenase subunit in IFM, but not in SSM. Because of these bidirectional changes, the average overall half-life of proteins was not altered by heart failure in both SSM and IFM. The kinetic measurements of individual mitochondrial proteins presented in this study may contribute to a better understanding of the mechanisms responsible for mitochondrial alterations in the failing heart.
Journal of Molecular and Cellular Cardiology 07/2014; · 5.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Imatinib mesylate is a selective tyrosine-kinase inhibitor used in the treatment of multiple cancers, most notably chronic myelogenous leukemia. There is evidence that imatinib can induce cardiotoxicity in cancer patients. Our hypothesis is that imatinib alters calcium regulatory mechanisms and can contribute to development of pathological cardiac hypertrophy.
Clinical and Translational Science 06/2014; · 2.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: -The gut-derived hormone ghrelin, especially its acylated form, plays a major role in the regulation of systemic metabolism and exerts also relevant cardioprotective effects, hence it has been proposed for the treatment of heart failure (HF). We tested the hypothesis that ghrelin can directly modulate cardiac energy substrate metabolism.
[Show abstract][Hide abstract] ABSTRACT: Myocardial hibernation (MH) is a well-known feature of human ischaemic cardiomyopathy (ICM), whereas its presence in human idiopathic dilated cardiomyopathy (DCM) is still controversial. We investigated the histological and molecular features of MH in left ventricle (LV) regions of failing DCM or ICM hearts. We examined failing hearts from DCM (n = 11; 41.9 ± 5.45 years; left ventricle-ejection fraction (LV-EF), 18 ± 3.16%) and ICM patients (n = 12; 58.08 ± 1.7 years; LVEF, 21.5 ± 6.08%) undergoing cardiac transplantation, and normal donor hearts (N, n = 8). LV inter-ventricular septum (IVS) and antero-lateral free wall (FW) were transmurally (i.e. sub-epicardial, mesocardial and sub-endocardial layers) analysed. LV glycogen content was shown to be increased in both DCM and ICM as compared with N hearts (P < 0.001), with a U-shaped transmural distribution (lower values in mesocardium). Capillary density was homogenously reduced in both DCM and ICM as compared with N (P < 0.05 versus N), with a lower decrease independent of the extent of fibrosis in sub-endocardial and sub-epicardial layers of DCM as compared with ICM. HIF1-α and nestin, recognized ischaemic molecular hallmarks, were similarly expressed in DCM-LV and ICM-LV myocardium. The proteomic profile was overlapping by ~50% in DCM and ICM groups. Morphological and molecular features of MH were detected in end-stage ICM as well as in end-stage DCM LV, despite epicardial coronary artery patency and lower fibrosis in DCM hearts. Unravelling the presence of MH in the absence of coronary stenosis may be helpful to design a novel approach in the clinical management of DCM.
Journal of Cellular and Molecular Medicine 01/2014; · 4.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In vitro studies suggested that glucose metabolism through the oxidative pentose phosphate pathway (oxPPP) can paradoxically feed superoxide-generating enzymes in failing hearts. We therefore tested the hypothesis that acute inhibition of the oxPPP reduces oxidative stress and enhances function and metabolism of the failing heart, in vivo. In 10 chronically instrumented dogs, congestive heart failure (HF) was induced by high-frequency cardiac pacing. Myocardial glucose consumption was enhanced by raising arterial glycemia to levels mimicking post-prandial peaks, before and after intravenous administration of the oxPPP inhibitor 6-aminonicotinamide (80 mg/kg). Myocardial energy substrate metabolism was measured with radiolabeled glucose and oleic acid, and cardiac 8-isoprostane output was used as an index of oxidative stress. A group of 5 chronically instrumented, normal dogs served as control. In HF, raising glycemic levels from ~80 to ~170 mg/dL increased cardiac isoprostane output by approximately 2-fold, while oxPPP inhibition normalized oxidative stress and enhanced cardiac oxygen consumption, glucose oxidation and stroke work. In normal hearts glucose infusion did not induce significant changes in cardiac oxidative stress. Myocardial tissue concentration of 6P-gluconate, an intermediate metabolite of the oxPPP, was significantly reduced by approximately 50% in treated vs non-treated failing hearts, supporting the inhibitory effect of 6-aminonicotinamide. Our study indicates an important contribution of the oxPPP activity to cardiac oxidative stress in HF, which is particularly pronounced during common physiological changes such as post-prandial glycemic peaks.
AJP Heart and Circulatory Physiology 01/2014; · 4.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nanolithography techniques enable the fabrication of complex nanodevices that can be used for biosensing purposes. However, these devices are normally supported by a substrate and their use is limited to in vitro applications. Following a top-down procedure, we designed and fabricated composite inductance-capacitance (LC) nanoresonators that can be detached from their substrate and dispersed in water. The multimaterial composition of these resonators makes it possible to differentially functionalize different parts of the device to obtain stable aqueous suspensions and multi-sensing capabilities. For the first time, we demonstrate detection of these devices in an aqueous environment, and we show that they can be sensitized to their local environment and to chemical binding of specific molecular moieties. The possibility to optically probe the nanoresonator resonance in liquid dispersions paves the way to a variety of new applications, including injection into living organisms for in vivo sensing and imaging.
PLoS ONE 01/2014; 9(8):e105474. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Apoptosis is a key determinant of major pathological processes, including chronic cardiac failure. We developed and tested in vitro a novel system to induce cardiomyocyte-specific apoptosis by virus-mediated delivery of a conditional transgene. The entire system was packaged in a lentiviral vector. We used the cardiomyocyte-specific Na(+)-Ca(2+) exchange promoter to control the transcription of the reverse tetracycline transactivator, while the transgene expression was driven by the tetracycline-responsive element. The proapoptotic transgene of choice was the short isoform of the apoptosis-inducing factor, known to quickly induce the caspase-independent apoptosis when overexpressed in cells. Transduction of cardiomyocyte cells with this vector caused a tetracycline-regulated induction of apoptosis, which was not observed in noncardiac cells. Therefore, our system proved a valuable molecular tool for inducing controlled apoptosis in selected cells. Furthermore, the vector we developed is suitable for "lentivirus transgenesis," an interesting strategy recently proposed for the genetic manipulation of animals other than mice, including large mammals.
BioMed research international. 01/2013; 2013:845816.
[Show abstract][Hide abstract] ABSTRACT: Glucose 6-phosphate dehydrogenase (G6PD) catalyzes the rate-determining step in the pentose phosphate pathway, and produces NADPH to fuel glutathione recycling. G6PD deficiency is the most common enzyme deficiency in humans, and affects over 400 million people worldwide; however its impact on cardiovascular disease is poorly understood. The glutathione pathway is paramount to antioxidant defense, and G6PD deficient cells do not cope well with oxidative damage. Limited clinical evidence indicates that G6PD deficiency may be associated with hypertension. However, there is also data to support a protective role of G6PD deficiency in decreasing the risk of heart disease and cardiovascular-associated deaths, perhaps through a decrease in cholesterol synthesis. Studies in G6PD deficient (G6PDX) mice are mixed, and provide evidence for both protective and deleterious effects. G6PD deficiency may provide a protective effect through decreasing cholesterol synthesis, superoxide production, and reductive stress. However, recent studies indicate that G6PDX mice are moderately more susceptible to ventricular dilation in response to myocardial infarction or pressure overload-induced heart failure. Further, G6PDX hearts do not recover as well as non-deficient mice when faced with ischemia-reperfusion injury, and G6PDX mice are susceptible to the development of age-associated cardiac hypertrophy. Overall, the limited available data indicate a complex interplay in which adverse effects of G6PD deficiency may outweigh potential protective effects in the face of cardiac stress. Definitive clinical studies in large populations are needed to determine the effects of G6PD deficiency on the development of cardiovascular disease and subsequent outcomes.
AJP Heart and Circulatory Physiology 12/2012; · 4.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: -Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency in the world. In failing hearts, G6PD is upregulated and generates NADPH that is used by the glutathione pathway to remove reactive oxygen species (ROS), but also as a substrate by ROS-generating enzymes. Therefore, G6PD deficiency might prevent heart failure by decreasing NADPH and ROS production. METHODS AND RESULTS: -This hypothesis was evaluated in a mouse model of human G6PD deficiency (G6PDX mice, ~40% normal activity). Myocardial infarction with 3 months follow-up resulted in LV dilation and dysfunction in both WT and G6PDX mice, but significantly greater end diastolic volume and wall thinning in G6PDX mice. Similarly, pressure overload induced by transverse aortic constriction (TAC) for 6 weeks caused greater LV dilation in G6PDX mice than WT. We further stressed TAC mice by feeding a high fructose diet to increase flux through G6PD and ROS production, and again observed worse LV remodeling and a lower ejection fraction in G6PDX than WT mice. Tissue content of lipid peroxidation products was increased in G6PDX mice in response to infarction and aconitase activity was decreased with TAC, suggesting that G6PD deficiency increases myocardial oxidative stress and subsequent damage. CONCLUSIONS: -Contrary to our hypothesis, G6PD deficiency increased redox stress in response to infarction or pressure overload. However, we found only a modest acceleration of LV remodeling, suggesting that, in individuals with G6PD deficiency and concurrent hypertension or myocardial infarction, the risk for developing heart failure is higher, but limited by compensatory mechanisms.
[Show abstract][Hide abstract] ABSTRACT: The effects of vagal stimulation (VS) on cardiac energy substrate metabolism are unknown. We tested the hypothesis that acute VS alters the balance between free fatty acid (FFA) and carbohydrate oxidation and opposes the metabolic effects of beta-adrenergic stimulation. A clinical-type selective stimulator of the vagal efferent fibers was connected to the intact right vagus in chronically instrumented dogs. VS was set to reduce heart rate by 30 beats min-1, then the confounding effects of bradycardia were eliminated by pacing the heart at 165 beats min-1. 3H-oleate and 14C-glucose were infused to measure FFA and glucose oxidation. The heart was subjected to beta-adrenergic stress by infusing 5, 10 and 15 µg kg-1 min-1 of dobutamine before and during VS. We found that VS did not significantly affect baseline cardiac performance, haemodynamics and myocardial metabolism. However, at peak dobutamine stress, VS attenuated the increase in left ventricular pressure-diameter area from 235.9±72.8% to 167.3±55.8%, and in cardiac oxygen consumption from 173.9±23.3% to 127.89±6.2% (both P<0.05), thus mechanical efficiency was not enhanced. The increase in glucose oxidation fell from 289.3%±55.5 to 131.1±20.9 (both P<0.05), while FFA oxidation was not increased by beta-adrenergic stress and fell below baseline during VS only at the lowest dose of dobutamine. The functional and in part the metabolic changes were reversed by 0.1 mg kg-1 atropine I.V. Our data show that acute right VS does not affect baseline cardiac metabolism, but attenuates myocardial oxygen consumption and glucose oxidation in response adrenergic stress, thus functioning as a cardio-selective antagonist to beta-adrenergic activation.
The Journal of Physiology 09/2012; · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We tested the hypothesis that myocardial contractile protein phosphorylation and the Ca(2+) sensitivity of force production are dysregulated in a porcine model of pacing-induced heart failure (HF). The level of protein kinase A (PKA)-dependent cardiac troponin I (TnI) phosphorylation was lower in the myocardium surrounding the pacing electrode (pacing site) of the failing left ventricle (LV) than in the controls. Immunohistochemical assays of the LV pacing site pointed to isolated clusters of cardiomyocytes exhibiting a reduced level of phosphorylated TnI. Flow cytometry on isolated and permeabilized cardiomyocytes revealed a significantly larger cell-to-cell variation in the level of TnI phosphorylation of the LV pacing site than in the opposite region in HF or in either region in the controls: the interquartile range (IQR) on the distribution histogram of relative TnI phosphorylation was wider at the pacing site (IQR = 0.53) than that at the remote site of HF (IQR = 0.42; P = 0.0047) or that of the free wall of the control animals (IQR = 0.36; P = 0.0093). Additionally, the Ca(2+) sensitivities of isometric force production were higher and appeared to be more variable in single permeabilized cardiomyocytes from the HF pacing site than in the healthy myocardium. In conclusion, the level of PKA-dependent TnI phosphorylation and the Ca(2+) sensitivity of force production exhibited a high cell-to-cell variability at the LV pacing site, possibly explaining the abnormalities of the regional myocardial contractile function in a porcine model of pacing-induced HF.
Archiv für Kreislaufforschung 03/2012; 107(2):244. · 7.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The present study was conducted to test predictions of the oxidative stress theory of aging assessing reactive oxygen species production and oxidative stress resistance in cultured fibroblasts from 13 primate species ranging in body size from 0.25 to 120 kg and in longevity from 20 to 90 years. We assessed both basal and stress-induced reactive oxygen species production in fibroblasts from five great apes (human, chimpanzee, bonobo, gorilla, and orangutan), four Old World monkeys (baboon, rhesus and crested black macaques, and patas monkey), three New World monkeys (common marmoset, red-bellied tamarin, and woolly monkey), and one lemur (ring-tailed lemur). Measurements of cellular MitoSox fluorescence, an indicator of mitochondrial superoxide (O2(·-)) generation, showed an inverse correlation between longevity and steady state or metabolic stress-induced mitochondrial O2(·-) production, but this correlation was lost when the effects of body mass were removed, and the data were analyzed using phylogenetically independent contrasts. Fibroblasts from longer-lived primate species also exhibited superior resistance to H(2)O(2)-induced apoptotic cell death than cells from shorter-living primates. After correction for body mass and lack of phylogenetic independence, this correlation, although still discernible, fell short of significance by regression analysis. Thus, increased longevity in this sample of primates is not causally associated with low cellular reactive oxygen species generation, but further studies are warranted to test the association between increased cellular resistance to oxidative stressor and primate longevity.
The Journals of Gerontology Series A Biological Sciences and Medical Sciences 01/2012; 67(8):841-52. · 4.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hyperpolarization of ¹³C-labeled energy substrates enables the noninvasive detection and mapping of metabolic activity, in vivo, with magnetic resonance spectroscopy (MRS). Therefore, hyperpolarization and ¹³C MRS can potentially become a powerful tool to study the physiology of organs such as the heart, through the quantification of kinetic patterns under both normal and pathological conditions. In this study we assessed myocardial uptake and metabolism of hyperpolarized [1-¹³C]pyruvate in anesthetized pigs. Pyruvate metabolism was studied at baseline and during dobutamine-induced stimulation. We applied a numerical approach for spectral analysis and kinetic fitting (LSFIT/KIMOfit), making a comparison with a well-known jMRUI/AMARES analysis and γ-variate function, and we estimated the apparent conversion rate of hyperpolarized [1-¹³C]pyruvate into its downstream metabolites [1-¹³C]lactate, [1-¹³C]alanine and [¹³C]bicarbonate in a 3 T MR scanner. We detected an increase in the apparent kinetic constants (k(PX) ) for bicarbonate and lactate of two-fold during dobutamine infusion. These data correlate with the double product (rate-pressure product), an indirect parameter of cardiac oxygen consumption: we observed an increase in value by 46 ± 11% during inotropic stress. The proposed approach might be applied to future studies in models of cardiac disease and/or for the assessment of the pharmacokinetic properties of suitable ¹³C-enriched tracers for MRS.
[Show abstract][Hide abstract] ABSTRACT: Stem cells possess the ability to terminally differentiate in cell phenotypes belonging to several different lineages. Over the last decade, transplant of adult stem cells into the injuried myocardium has been widely studied as a revolutionary approach to promote the non-pharmacological improvement or replacement of the lost function. In spite of the tantalizing perspectives and controversial results, several questions about the viability and biology of transplanted stem cells in the beating heart still remain unanswered, mostly because of the current technological limitations. Recent advances in bio- and nano-technology are allowing the development of molecular probes for imaging thus providing a better understanding of stem cells physiology and fate in vivo. Reporter gene based molecular imaging is a high-throughput and sensitive tool used to unscramble over time the mechanisms underlying cell-induced myocardial repair in vivo. To date, the employed reporter genes have been exogenous (proteins which are expressed after gene engineering), or endogenous (detected by tracer substrates). This review will highlight current and outstanding experimental investigations, which are developing new probes to monitor the fate of stem cells transplanted in failing myocardium in vivo.