A comparison of the genetic activity of pyrvinium pamoate with that of several other anthelmintic drugs in Saccharomyces cerevisiae

Several anthelmintic drugs that are used routinely in oxyuriasis therapy were analyzed for genotoxicity in a diploid mitotic recombination and gene conversion assay (strain D5 of Saccharomyces cerevisiae), and in a haploid yeast reversion assay (strain XV185-14C). Piperazine citrate, piperazine adipate, mebendazole and thiabendazole did not appear to be genotoxic in either yeast strain. Pyrvinium pamoate induced the reversion of the missense, nonsense and frameshift alleles in strain XV185-14C, whereas pyrantel pamoate induced only the reversion of the frameshift allele. Pyrvinium pamoate was recombinogenic in strain D5, and there is an indication that pyrantel pamoate, at the lowest dose that was tested, might induce gene conversion or aneuploidy.     

Natural Aminoacyl tRNA Synthetase Fragment Enhances Cardiac Function after Myocardial Infarction

A naturally-occurring fragment of tyrosyl-tRNA synthetase (TyrRS) has been shown in higher eukaryotes to ‘moonlight’ as a pro-angiogenic cytokine in addition to its primary role in protein translation. Pro-angiogenic cytokines have previously been proposed to be promising therapeutic mechanisms for the treatment of myocardial infarction. Here, we show that systemic delivery of the natural fragment of TyRS, mini-TyrRS, improves heart function in mice after myocardial infarction. This improvement is associated with reduced formation of scar tissue, increased angiogenesis of cardiac capillaries, recruitment of c-kit^pos cells and proliferation of myocardial fibroblasts. This work demonstrates that mini-TyrRS has beneficial effects on cardiac repair and regeneration and offers support for the notion that elucidation of the ever expanding repertoire of noncanonical functions of aminoacyl tRNA synthetases offers unique opportunities for development of novel therapeutics.     

Injection of mesenchymal stromal cells into a mechanically stimulated in vitro model of cardiac fibrosis has paracrine effects on resident fibroblasts

Background aimsMyocardial infarction results in the formation of scar tissue populated by myofibroblasts, a phenotype characterized by increased contractility and matrix deposition. Mesenchymal stromal cells (MSC) delivered to the myocardium can attenuate scar growth and restore cardiac function, though the mechanism is unclear.MethodsThis study describes a simple yet robust three-dimensional (3D) in vitro co-culture model to examine the paracrine effects of implanted MSC on resident myofibroblasts in a controlled biochemical and mechanical environment. The fibrosis model consisted of fibroblasts embedded in a 3D collagen gel cultured under defined oxygen tensions and exposed to either cyclic strain or interstitial fluid flow. MSC were injected into this model, and the effect on fibroblast phenotype was evaluated 48 h after cell injection.ResultsAnalysis of gene and protein expression of the fibroblasts indicated that injection of MSC attenuated the myofibroblast transition in response to reduced oxygen and mechanical stress. Assessment of vascular endothelial growth factor and insulin-like growth factor-1 levels demonstrated that their release by fibroblasts was markedly upregulated in hypoxic conditions but attenuated by strain or fluid flow. In fibroblast-MSC co-cultures, vascular endothelial growth factor levels were increased by hypoxia but not affected by mechanical stimuli, whereas insulin-like growth factor-1 levels were generally low and not affected by experimental conditions.ConclusionsThis study demonstrates how a 3D in vitro model of the cardiac scar can be used to examine paracrine effects of MSC on the phenotype of resident fibroblasts and therefore illuminates the role of injected progenitor cells on the progression of cardiac fibrosis.     

Knock‐out of MicroRNA 145 impairs cardiac fibroblast function and wound healing post‐myocardial infarction

Prevention of infarct scar thinning and dilatation and stimulation of scar contracture can prevent progressive heart failure. Since microRNA 145 (miR‐145) plays an important role in cardiac fibroblast response to wound healing and cardiac repair after an myocardial infarction (MI), using a miR‐145 knock‐out (KO) mouse model, we evaluated contribution of down‐regulation of miR‐145 to cardiac fibroblast and myofibroblast function during adverse cardiac remodelling. Cardiac function decreased more and the infarct size was larger in miR‐145 KO than that in WT mice after MI and this phenomenon was accompanied by a decrease in cardiac fibroblast‐to‐myofibroblast differentiation. Quantification of collagen I and α‐SMA protein levels as well as wound contraction revealed that transdifferentiation of cardiac fibroblasts into myofibroblasts was lower in KO than WT mice. In vitro restoration of miR‐145 induced more differentiation of fibroblasts to myofibroblasts and this effect involved the target genes Klf4 and myocardin. MiR‐145 contributes to infarct scar contraction in the heart and the absence of miR‐145 contributes to dysfunction of cardiac fibroblast, resulting in greater infarct thinning and dilatation. Augmentation of miR‐145 could be an attractive target to prevent adverse cardiac remodelling after MI by enhancing the phenotypic switch of cardiac fibroblasts to myofibroblasts.     

Cardiotrophin-1: Expression in experimental myocardial infarction and potential role in post-MI wound healing

Cardiotrophin-1 (CT-1), a member of the IL-6 family of cytokines, has been shown to be elevated in the serum of patients with ischemic heart disease and valvular heart disease, and induces cardiomyocyte hypertrophy in vitro. We investigated expression of CT-1 in post-MI rat heart and the effect of CT-1 on cultured primary adult rat cardiac fibroblasts. Elevated CT-1 expression was observed in the infarct zone at 24 h and continued through 2, 4 and 8 weeks post-MI, compared to sham-operated animals. CT-1 induced rapid phosphorylation of Jak1, Jak2, STAT1, STAT3, p42/44 MAPK and Akt in cultured adult cardiac fibroblasts. CT-1 induced cardiac fibroblast protein synthesis and proliferation. Protein and DNA synthesis were dependent on activation of Jak/STAT, MEK1/2, PI3K and Src pathways as evidenced by decreased ³H-leucine and ³H-thymidine incorporation after pretreatment with AG490, PD98059, LY294002 and genistein respectively. Furthermore, CT-1 treatment increased procollagen-1-carboxypropeptide (P1CP) synthesis, a marker of mature collagen synthesis. CT-1 induced cell migration of rat cardiac fibroblasts. Our results suggest that CT-1, as expressed in post-MI heart, may play an important role in infarct scar formation and ongoing remodeling of the scar. CT-1 was able to initiate each of the processes considered important in the formation of infarct scar including cardiac fibroblast migration as well as fibroblast proliferation and collagen synthesis. Further work is required to determine factors that induce CT-1 expression and interplay with other mediators of cardiac infarct wound healing in the setting of acute cardiac ischemia and chronic post-MI heart failure.     

Systematic Characterization of Myocardial Inflammation,Repair, and Remodeling in a Mouse Model of Reperfused Myocardial Infarction

Mouse models of myocardial infarction are essential tools for the study of cardiac injury, repair, and remodeling. Our current investigation establishes a systematic approach for quantitative evaluation of the inflammatory and reparative response, cardiac function, and geometry in a mouse model of reperfused myocardial infarction. Reperfused mouse infarcts exhibited marked induction of inflammatory cytokines that peaked after 6 hr of reperfusion. In the infarcted heart, scar contraction and chamber dilation continued for at least 28 days after reperfusion; infarct maturation was associated with marked thinning of the scar, accompanied by volume loss and rapid clearance of cellular elements. Echocardiographic measurements of end-diastolic dimensions correlated well with morphometric assessment of dilative remodeling in perfusion-fixed hearts. Hemodynamic monitoring was used to quantitatively assess systolic and diastolic function; the severity of diastolic dysfunction following myocardial infarction correlated with cardiomyocyte hypertrophy and infarct collagen content. Expression of molecular mediators of inflammation and cellular infiltration needs to be investigated during the first 72 hr, whereas assessment of dilative remodeling requires measurement of geometric parameters for at least four weeks after the acute event. Rapid initiation and resolution of the inflammatory response, accelerated scar maturation, and extensive infarct volume loss are important characteristics of infarct healing in mice.     

Myocardial postischemic injury is reduced by polyADPripose polymerase-1 gene disruption

BACKGROUND: PolyADPribose polymerase (PARP) is activated by DNA strand breaks to catalyze the addition of ADPribose groups to nuclear proteins, especially PARP-1. Excessive polyADPribosylation leads to cell death through depletion of NAD+ and ATP. MATERIALS AND METHODS: In vivo PARP activation in heart tissue slices was assayed through conversion of [33P]NAD+ into polyADPribose (PAR) following ischemia-reperfusion (I/R) and also monitored by immunohistochemical staining for PAR. Cardiac contractility, nitric oxide (NO), reactive oxygen species (ROS), NAD+ and ATP levels were examined in wild type (WT) and in PARP-1 gene-deleted (PARP-1(-/-)) isolated, perfused mouse hearts. Myocardial infarct size was assessed following coronary artery occlusion in rats treated with PARP inhibitors. RESULTS: Ischemia-reperfusion (I/R) augmented formation of nitric oxide, oxygen free radicals and PARP activity. I/R induced decreases in cardiac contractility and NAD+ levels were attenuated in PARP-1(-/-) mouse hearts. PARP inhibitors reduced myocardial infarct size in rats. Residual polyADPribosylation in PARP-1(-/-) hearts may reflect alternative forms of PARP. CONCLUSIONS: PolyADPribosylation from PARP-1 and other sources of enzymatic PAR synthesis is associated with cardiac damage following myocardial ischemia. PARP inhibitors may have therapeutic utility in myocardial disease.     

Myocardial Mitochondrial and Contractile Function Are Preserved in Mice Lacking Adiponectin

Adiponectin deficiency leads to increased myocardial infarct size following ischemia reperfusion and to exaggerated cardiac hypertrophy following pressure overload, entities that are causally linked to mitochondrial dysfunction. In skeletal muscle, lack of adiponectin results in impaired mitochondrial function. Thus, it was our objective to investigate whether adiponectin deficiency impairs mitochondrial energetics in the heart. At 8 weeks of age, heart weight-to-body weight ratios were not different between adiponectin knockout (ADQ^-/-) mice and wildtypes (WT). In isolated working hearts, cardiac output, aortic developed pressure and cardiac power were preserved in ADQ^-/- mice. Rates of fatty acid oxidation, glucose oxidation and glycolysis were unchanged between groups. While myocardial oxygen consumption was slightly reduced (-24%) in ADQ^-/- mice in isolated working hearts, rates of maximal ADP-stimulated mitochondrial oxygen consumption and ATP synthesis in saponin-permeabilized cardiac fibers were preserved in ADQ^-/- mice with glutamate, pyruvate or palmitoyl-carnitine as a substrate. In addition, enzymatic activity of respiratory complexes I and II was unchanged between groups. Phosphorylation of AMP-activated protein kinase and SIRT1 activity were not decreased, expression and acetylation of PGC-1α were unchanged, and mitochondrial content of OXPHOS subunits was not decreased in ADQ^-/- mice. Finally, increasing energy demands due to prolonged subcutaneous infusion of isoproterenol did not differentially affect cardiac contractility or mitochondrial function in ADQ^-/- mice compared to WT. Thus, mitochondrial and contractile function are preserved in hearts of mice lacking adiponectin, suggesting that adiponectin may be expendable in the regulation of mitochondrial energetics and contractile function in the heart under non-pathological conditions.     

Mesenchymal stem cells and the treatment of cardiac disease

The ischemia-induced death of cardiomyocytes results in scar formation and reduced contractility of the ventricle. Several preclinical and clinical studies have supported the notion that cell therapy may be used for cardiac regeneration. Most attempts for cardiomyoplasty have considered the bone marrow as the source of the "repair stem cell(s)," assuming that the hematopoietic stem cell can do the work. However, bone marrow is also the residence of other progenitor cells, including mesenchymal stem cells (MSCs). Since 1995 it has been known that under in vitro conditions, MSCs differentiate into cells exhibiting features of cardiomyocytes. This pioneer work was followed by many preclinical studies that revealed that ex vivo expanded, bone marrow-derived MSCs may represent another option for cardiac regeneration. In this work, we review evidence and new prospects that support the use of MSCs in cardiomyoplasty.     

Reprogramming a broken heart

Fibrosis resulting from cardiac injury presents a major challenge to restoring heart function after myocardial infarction. Two recent papers in Nature report successful in vivo reprogramming of fibroblasts to cardiomyocytes in injured mouse hearts (Qian et al., 2012; Song et al., 2012), resulting in improved cardiac function and reduced scar formation.     

运动后心力及心率同时遥测的心力恢复趋势与心率恢复趋势的对比研究

为了更加全面地研究和评估心肌的变时性和变力性,研制了能够在运动场地同时采集心力和心率的心音信号遥测系统。采集了50名体育系学生和30名其他系学生在完成规定运动量的登梯运动后,5min内连续变化的心音信号。对采集的数据经过3次样条插值、均匀采样和小波变换滤波后,得到了平滑的心力恢复趋势曲线和心率恢复趋势曲线。统计分析表明,大负荷运动量下的心脏储备主要来自于心力储备而不是心率储备(P<0.001),和其他系学生相比,体育系学生具有的高水平心功能不仅在于具有更大的心力储备指数(P<0.001),而且还在于具有更快的心力恢复速度(P<0.001)。同时检测心力恢复趋势与心率恢复趋势会有益于全面评估心脏功能。     

Cardioprotective effects of erythropoietin in the reperfused ischemic heart: a potential role for cardiac fibroblasts

Erythropoietin has recently been shown to have effects beyond hematopoiesis such as prevention of neuronal and cardiac apoptosis secondary to ischemia. In this study, we evaluated the in vivo protective potential of erythropoietin in the reperfused rabbit heart following ventricular ischemia. We show that "preconditioning" with erythropoietin activates cell survival pathways in myocardial tissue in vivo and adult rabbit cardiac fibroblasts in vitro. These pathways, activated by erythropoietin in both whole hearts and cardiac fibroblasts, are also activated acutely by ischemia/reperfusion injury. Moreover, in vivo studies indicate that erythropoietin treatment either prior to or during ischemia significantly enhances cardiac function and recovery, including left ventricular contractility, following myocardial ischemia/reperfusion. Our data indicate that a contributing in vivo cellular mechanism of this protection is mitigation of myocardial cell apoptosis. This results in decreased infarct size as evidenced by area at risk studies following in vivo ischemia/reperfusion injury, translating into more viable myocardium and less ventricular dysfunction. Therefore, erythropoietin treatment may offer novel protection against ischemic heart disease and may act, at least in part, by direct action on cardiac fibroblasts and myocytes to alter survival and ventricular remodeling.     

Pyrvinium, a potent small molecule Wnt inhibitor, promotes wound repair and post-MI cardiac remodeling

Wnt signaling plays an important role in developmental and stem cell biology. To test the hypothesis that temporary inhibition of Wnt signaling will enhance granulation tissue and promote angiogenesis in tissue repair, we employed a recently characterized small molecule Wnt inhibitor. Pyrvinium is an FDA-approved drug that we identified as a Wnt inhibitor in a chemical screen for small molecules that stabilize β-catenin and inhibit Axin degradation. Our subsequent characterization of pyrvinium has revealed that its critical cellular target in the Wnt pathway is Casein Kinase 1α. Daily administration of pyrvinium directly into polyvinyl alcohol (PVA) sponges implanted subcutaneously in mice generated better organized and vascularized granulation tissue; this compound also increased the proliferative index of the tissue within the sponges. To evaluate its effect in myocardial repair, we induced a myocardial infarction (MI) by coronary artery ligation and administered a single intramyocardial dose of pyrvinium. Mice were evaluated by echocardiography at 7 and 30 days post-MI and treatment; post mortem hearts were evaluated by histology at 30 days. Pyrvinium reduced adverse cardiac remodeling demonstrated by decreased left ventricular internal diameter in diastole (LVIDD) as compared to a control compound. Increased Ki-67+ cells were observed in peri-infarct and distal myocardium of pyrvinium-treated animals. These results need to be further followed-up to determine if therapeutic inhibition of canonical Wnt may avert adverse remodeling after ischemic injury and its impact on myocardial repair and regeneration.     

The actions of insulin on cardiac contractility

This review summarizes the effects of insulin on cardiac contractility of the normal, failing, anoxic and ischemic heart. In animal experiments under aerobic conditions a variety of effects of insulin on cardiac contractile force have been described which are frequently contradictory. The use of different insulin preparations of unknown purity make the interpretation of these findings rather difficult. The protective effects of insulin in the isolated heart on the rate of production of spontaneous heart failure require both glucose and insulin for maximal effect and are probably related to an improved cardiac carbohydrate utilization produced by insulin. Other cardiac effects of insulin independent of glucose could be due to ionic readjustments produced by insulin which are glucose independent.     

An anticancer agent, pyrvinium pamoate inhibits the NADH-fumarate reductase system--a unique mitochondrial energy metabolism in tumour microenvironments

Increased glycolysis is the principal explanation for how cancer cells generate energy in the absence of oxygen. However, in actual human tumour microenvironments, hypoxia is often associated with hypoglycemia because of the poor blood supply. Therefore, glycolysis cannot be the sole mechanism for the maintenance of the energy status in cancers. To understand energy metabolism in cancer cells under hypoxia-hypoglycemic conditions mimicking the tumour microenvironments, we examined the NADH-fumarate reductase (NADH-FR) system, which functions in parasites under hypoxic condition, as a candidate mechanism. In human cancer cells (DLD-1, Panc-1 and HepG2) cultured under hypoxic-hypoglycemic conditions, NADH-FR activity, which is composed of the activities of complex I (NADH-ubiquinone reductase) and the reverse reaction of complex II (quinol-FR), increased, whereas NADH-oxidase activity decreased. Pyrvinium pamoate (PP), which is an anthelmintic and has an anti-cancer effect within tumour-mimicking microenvironments, inhibited NADH-FR activities in both parasites and mammalian mitochondria. Moreover, PP increased the activity of complex II (succinate-ubiquinone reductase) in mitochondria from human cancer cells cultured under normoxia-normoglycemic conditions but not under hypoxia-hypoglycemic conditions. These results indicate that the NADH-FR system may be important for maintaining mitochondrial energy production in tumour microenvironments and suggest its potential use as a novel therapeutic target.     

Effects of elevated glucose levels on interactions of cardiac fibroblasts with the extracellular matrix

Exposure of fibroblasts to high glucose levels promotes a fibrotic response characterized by increased expression of extracellular matrix components including interstitial collagens. Little is known about the effects of glucose levels on other aspects of fibroblast function. Fibroblasts in the myocardium are surrounded by an extensive extracellular matrix composed predominantly of type I collagen. Interactions between fibroblasts and the myocardial extracellular matrix are thought to affect heart function by altering ventricular diastolic properties. The purpose of the present study was to determine the effects of elevated glucose levels on the interactions between heart fibroblasts and the collagenous extracellular matrix. Studies were performed to determine the effects of relative glucose levels on the ability of fibroblasts to migrate on and contract a three-dimensional collagenous substratum. These experiments illustrated that exposure of cardiac fibroblasts to high glucose levels (25 mM) resulted in decreased migratory activity of fibroblasts on a collagen matrix and decreased fibroblast proliferation. In addition, high glucose stimulated collagen and collagen-binding integrin expression and contraction of three-dimensional collagen gels by cardiac fibroblasts. These studies illustrate that altered glucose levels induce important changes in the interactions of cardiac fibroblasts with the collagenous extracellular matrix. Xiaoyi Zhang and James A. Stewart, Jr. are co-first authors.     

Adaptive mechanisms to compensate for overnutrition-induced cardiovascular abnormalities

In conditions of overnutrition, cardiac cells must cope with a multitude of extracellular signals generated by changes in nutrient load (glucose, amino acids, and lipids) and the hormonal milieu [increased insulin (INS), ANG II, and adverse cytokine/adipokine profile]. Herein, we review the diverse compensatory/adaptive mechanisms that counter the deleterious effects of excess nutrients and growth factors. We largely focus the discussion on evidence obtained from Zucker obese (ZO) and Zucker diabetic fatty (ZDF) rats, which are useful models to evaluate adaptive and maladaptive metabolic, structural, and functional cardiac remodeling. One adaptive mechanism present in the INS-resistant ZO, but absent in the diabetic ZDF heart, involves an interaction between the nutrient sensor kinase mammalian target of rapamycin complex 1 (mTORC1) and ANG II-type 2 receptor (AT2R). Recent evidence supports a cardioprotective role for the AT2R; for example, suppression of AT2R activation interferes with antihypertrophic/antifibrotic effects of AT1R blockade, and AT2R agonism improves cardiac structure and function. We propose a scenario, whereby mTORC1-signaling-mediated increase in AT2R expression in the INS-resistant ZO heart is a cardioprotective adaptation to overnutrition. In contrast to the ZO rat, heart tissues of ZDF rats do not show activation of mTORC1. We posit that such a lack of activation of the mTOR?AT2R integrative pathway in cardiac tissue under conditions of obesity-induced diabetes may be a metabolic switch associated with INS deficiency and clinical diabetes.     

Control of doxorubicin-induced,reactive oxygen-related apoptosis by glutathione peroxidase 1 in cardiac fibroblasts

Reactive oxygen formation plays a mechanistic role in the cardiotoxicity of doxorubicin, a chemotherapeutic agent that remains an important component of treatment programs for breast cancer and hematopoietic malignancies. To examine the role of doxorubicin-induced reactive oxygen species (ROS) in drug-related cardiac apoptosis, murine embryonic fibroblast cell lines were derived from the hearts of glutathione peroxidase 1 (Gpx-1) knockout mice. Cells from homozygous Gpx-1 knockout mice and parental animals were propagated with (Se+) and without (Se-) 100 nM sodium selenite. Activity levels of the peroxide detoxifying selenoprotein glutathione peroxidase (GSHPx) were marginally detectable (<1.6 nmol/min/mg) in fibroblasts from homozygous knockout animals whether or not cells were supplemented with selenium. GSHPx activity in Se- cells from parental murine fibroblasts was also <1.6 nmol/min/mg, whereas GSHPx levels in Se+ parental murine fibroblasts were 12.9 ± 2.7 nmol/min/mg (mean ± SE; P < 0.05). Catalase, superoxide dismutase, glutathione reductase, glutathione S-transferase, glucose 6-phosphate dehydrogenase, and reduced glutathione activities did not differ amongst the four cell lines. Reactive oxygen production increased from 908 ± 122 (arbitrary units) for untreated control cells to 1668 ± 54 following exposure to 1 μM doxorubicin for 24 h in parental fibroblasts not supplemented with selenium (P < 0.03); reactive oxygen formation in doxorubicin-treated parental fibroblasts propagated in selenium was 996 ± 69 (P = not significant compared to untreated control cells). Reactive oxygen levels in homozygous Gpx-1 knockout fibroblasts, irrespective of selenium supplementation status, were increased and equivalent to that in selenium deficient wild type fibroblasts. When cardiac fibroblasts were exposed to doxorubicin (0.05 μM) for 96 h and examined for cell cycle alterations by flow cytometry, and apoptosis by TUNEL assay, marked G2 arrest and TUNEL positivity were observed in knockout fibroblasts in the presence or absence of supplemental selenium, and in parental fibroblasts propagated without selenium. Parental fibroblasts propagated with selenium and exposed to the same concentration of doxorubicin demonstrated modest TUNEL positivity and substantially diminished amounts of low molecular weight DNA. These results were replicated in cardiac fibroblasts exposed to doxorubicin (1–2 μM) for 2 h (to mimic clinical drug dosing schedules) and examined 96 h following initiation of drug exposure. Doxorubicin uptake in cardiac fibroblasts was similar irrespective of the mRNA expression level or activity of GSHPx. These experiments suggest that the intracellular levels of doxorubicin-induced reactive oxygen species (ROS) are modulated by GSHPx and play an important role in doxorubicin-related apoptosis and altered cell cycle progression in murine cardiac fibroblasts.     

Ketone bodies alter dinitrophenol-induced glucose uptake through AMPK inhibition and oxidative stress generation in adult cardiomyocytes

In aerobic conditions, the heart preferentially oxidizes fatty acids. However, during metabolic stress, glucose becomes the major energy source, and enhanced glucose uptake has a protective effect on heart function and cardiomyocyte survival. Thus abnormal regulation of glucose uptake may contribute to the development of cardiac disease in diabetics. Ketone bodies are often elevated in poorly controlled diabetics and are associated with increased cellular oxidative stress. Thus we sought to determine the effect of the ketone body beta-hydroxybutyrate (OHB) on cardiac glucose uptake during metabolic stress. We used 2,4-dinitrophenol (DNP), an uncoupler of the mitochondrial oxidative chain, to mimic hypoxia in cardiomyocytes. Our data demonstrated that chronic exposure to OHB provoked a concentration-dependent decrease of DNP action, resulting in 56% inhibition of DNP-mediated glucose uptake at 5 mM OHB. This was paralleled by a diminution of DNP-mediated AMP-activated protein kinase (AMPK) and p38 MAPK phosphorylation. Chronic exposure to OHB also increased reactive oxygen species (ROS) production by 1.9-fold compared with control cells. To further understand the role of ROS in OHB action, cardiomyocytes were incubated with H(2)O(2). Our results demonstrated that this treatment diminished DNP-induced glucose uptake without altering activation of the AMPK/p38 MAPK signaling pathway. Incubation with the antioxidant N-acetylcysteine partially restored DNP-mediated glucose but not AMPK/p38 MAPK activation. In conclusion, these results suggest that ketone bodies, through inhibition of the AMPK/p38 MAPK signaling pathway and ROS overproduction, regulate DNP action and thus cardiac glucose uptake. Altered glucose uptake in hyperketonemic states during metabolic stress may contribute to diabetic cardiomyopathy.     

Bcl-2 is a key factor for cardiac fibroblast resistance to programmed cell death

Cardiac fibroblasts play an essential role in the physiology of the heart. These produce extracellular matrix proteins and synthesize angiogenic and cardioprotective factors. Although fibroblasts of cardiac origin are known to be resistant to apoptosis and to remain metabolically active in situations compromising cell survival, the underlying mechanisms are unknown. Here, we report that cardiac fibroblasts were more resistant than dermal or pulmonary fibroblasts to mitochondria-dependent cell death. Cytochrome c release was blocked in cardiac fibroblasts but not in dermal fibroblasts treated with staurosporine, etoposide, serum deprivation, or simulated ischemia, precluding caspase-3 activation and DNA fragmentation. Resistance to apoptosis of cardiac fibroblasts correlated with the expression of the anti-apoptotic protein Bcl-2, whereas skin and lung fibroblasts did not express detectable levels of this protein. Bcl-x(L,) Bax, and Bak were expressed at similar levels in cardiac, dermal, and lung fibroblasts. In addition, the death of cardiac fibroblasts during hypoxia was not associated with the cleavage of Bid but rather with Bcl-2 disappearance, suggesting the requirement of the mitochondrial apoptotic machinery to execute death receptor-induced programmed cell death. Knockdown of bcl-2 expression by siRNA in cardiac fibroblasts increased their apoptotic response to staurosporine, serum, and glucose deprivation and to simulated ischemia. Moreover, dermal fibroblasts overexpressing Bcl-2 achieved a similar level of resistance to these stimuli as cardiac fibroblasts. Thus, our data demonstrate that Bcl-2 is an important effector of heart fibroblast resistance to apoptosis and highlight a probable mechanism for promoting survival advantage in fibroblasts of cardiac origin.