Carmine Morisco

Akt Mediates the Cross-Talk Between β-Adrenergic and Insulin Receptors in Neonatal Cardiomyocytes

Upregulation of the sympathetic nervous system plays a key role in the pathogenesis of insulin resistance. Although the heart is a target organ of insulin, few studies have examined the mechanisms by which &bgr;-adrenergic stimulation affects insulin sensitivity in cardiac muscle. In this study, we explored the molecular mechanisms involved in the regulation of the cross-talk between &bgr; adrenergic and insulin receptors in neonatal rat cardiomyocytes and in transgenic mice with cardiac overexpression of a constitutively active mutant of Akt (E40K Tg). The results of this study show that &bgr;-adrenergic receptor stimulation has a biphasic effect on insulin-stimulated glucose uptake. Short-term stimulation induces an additive effect on insulin-induced glucose uptake, and this effect is mediated by phosphorylation of Akt in threonine 308 through PKA/Ca2+-dependent and PI3K-independent pathway, whereas insulin-evoked threonine phosphorylation of Akt is exclusively PI3K-dependent. On the other hand, long-term stimulation of &bgr;-adrenergic receptors inhibits both insulin-stimulated glucose uptake and insulin-induced autophosphorylation of the insulin receptor, and at the same time promotes threonine phosphorylation of the insulin receptor. This is mediated by serine 473 phosphorylation of Akt through PKA/Ca2+ and PI3K-dependent pathways. Under basal conditions, E40K Tg mice show increased levels of threonine phosphorylation of the &bgr; subunit of the insulin receptor and blunted tyrosine autophosphorylation of the &bgr;-subunit of the insulin receptor after insulin stimulation. These results indicate that, in cardiomyocytes, &bgr;-adrenergic receptor stimulation impairs insulin signaling transduction machinery through an Akt-dependent pathway, suggesting that Akt is critically involved in the regulation of insulin sensitivity.

Haploinsufficiency of the Hmga1 gene causes cardiac hypertrophy and myelo-lymphoproliferative disorders in mice.

The HMGA1 protein is a major factor in chromatin architecture and gene control. It plays a critical role in neoplastic transformation. In fact, blockage of HMGA1 synthesis prevents rat thyroid cell transformation by murine transforming retroviruses, and an adenovirus carrying the HMGA1 gene in the antisense orientation induces apoptotic cell death in anaplastic human thyroid carcinoma cell lines, but not in normal thyroid cells. Moreover, both in vitro and in vivo studies have established the oncogenic role of the HMGA1 gene. In this study, to define HMGA1 function in vivo, we examined the consequences of disrupting the Hmga1 gene in mice. Both heterozygous and homozygous mice for the Hmga1-null allele show cardiac hypertrophy due to the direct role of HMGA1 on cardiomyocytic cell growth regulation. These mice also developed hematologic malignancies, including B cell lymphoma and myeloid granuloerythroblastic leukemia. The B cell expansion and the increased expression of the RAG1/2 endonuclease, observed in HMGA1-knockout spleen tissues, might be responsible for the high rate of abnormal IgH rearrangements observed in these neoplasias. Therefore, the data reported here indicate the critical role of HMGA1 in heart development and growth, and reveal an unsuspected antioncogenic potential for this gene in hematologic malignancies.

Systemic hypertension and coronary artery disease: The link

A direct, continuous, and independent association between blood pressure values and incidence of coronary artery disease has been well documented. However, the evidence that the reduction of blood pressure alone is not able to completely reverse the increase in the risk of coronary artery disease associated with essential hypertension suggests that the link between hypertension and coronary artery disease is a complex process including other factors beside the increase in blood pressure values. In this regard, the main determinant of coronary artery disease in hypertensive patients seems to be the development of left ventricular hypertrophy (LVH). In fact, hypertensive patients who died from sudden cardiac death showed a lesser degree of coronary atherosclerosis compared with normotensives, but a higher incidence of LVH. Several mechanisms can account for the increased coronary risk with LVH, including (1) an increase in left ventricular (LV) mass, which by itself requires more oxygen for tissue perfusion; (2) impairment of coronary flow reserve; (3) perivascular fibrosis, which then impairs oxygen supply to the myocardium; and (4) deterioration of LV diastolic function, which hampers myocardial perfusion. Recently, a study reported an impairment of endothelial function and abnormal control of the sympathetic tone in hypertensive patients, which may contribute to the risk of coronary artery disease. In particular, the impaired endothelial function resulting in a prevalence of vasoconstrictive, thrombogenic, and proliferative factors may account for the enhanced ischemic susceptibility of these patients. Furthermore, the cardiac adrenergic system plays an important role in regulating myocardial blood flow. On one hand, hypertensive patients show an exaggerated sympathetic response to physiologic stimuli, whereas on the other hand, the beta-adrenergic receptor-mediated vasodilating component of the sympathetic response is blunted in hypertension. Finally, excess body weight, dyslipidemia, glucose intolerance, and hyperinsulinemia, which are frequently interrelated, represent independent predictors of both coronary artery disease and hypertension.

The use of a telematic connection for the follow-up of hypertensive patients improves the cardiovascular prognosis

Background Inadequate blood pressure (BP) control could be due to incorrect management of hypertensives caused by the lack of interaction between general practitioners (GP) and hypertension specialists. Objectives To test the effectiveness on BP and total cardiovascular risk (TCVR) control of an internet-based digital network connecting specialists and GPs. Methods We created a network among the Hypertension Clinic, Federico II University (Naples, Italy), 23 hospital-based hypertension clinics and 60 GPs from the area (CampaniaSalute Network, CS). Randomized GPs enrolled in CS could update online records of patients (n = 1979). As a control, we included 2045 patients referred to the specialist clinics by GPs from outside the network. All patients completed a 2-year follow-up. Results CS provided a larger reduction in BP [systolic/diastolic BP (SBP/DBP): 7.3 ± 0.4/5.4 ± 0.3 versus 4.1 ± 0.4/3.1 ± 0.26 mmHg, CS versus control; P < 0.001 for both] and percentage of patients with BP < 140/90 mmHg (CS versus control: baseline, 33 versus 34%, NS; end of follow-up, 51 versus 47%, χ2 = 13.371; P < 0.001). A European Society of Hypertension–European Society of Cardiology (ESH/ESC) TCVR score was calculated [from 1 (average) to 5 (very high TCVR)]. The CS group showed a reduction in the mean TCVR score (CS: from 3.5 ± 0.02 to 3.2 ± 0, P < 0.01, ANOVA; control group: 3.5 ± 0.03 to 3.4 ± 0.03, NS) and, accordingly, fatal and non-fatal major cardiovascular events (MACE) were less frequent (2.9 versus 4.3%; χ2 = 5.047, P < 0.02). CS predicts fewer MACE in multiple binary regression analysis (β:−7.27, P < 0.008) reducing the risk for MACE compared to control [odds ratio (OR): 0.838; 95% confidence interval (CI): 0.73–0.96]. Conclusion Our results support the idea that telemedicine can achieve better control of BP and TCVR.

TNF-α signal transduction in rat neonatal cardiac myocytes: definition of pathways generating from the TNF-α receptor

Cardiomyocyte hypertrophy and apoptosis have been implicated in the loss of contractile function during heart failure (HF). Moreover, patients with HF have been shown to exhibit increased levels of tumor necrosis factor a (TNF‐a) in the myocardium. However, the multiple signal transduction pathways generating from the TNF‐α receptor in cardiomyocytes and leading preferentially to apoptosis or hypertrophy are still unknown. Here we demonstrate in neonatal rat cardiomyocytes that 1) TNF‐α induces phosphorylation of AKT, activation of NF‐κB, and the phosphorylation of JUN kinase; 2) blocking AKT activity prevents NF‐κB activation, suggesting a role for AKT in regulating NF‐κB function; 3) AKT and JUN are both critical for the hypertrophic effects of TNF‐α, since dominant‐negative mutants of these genes are capable of inhibiting TNF‐α‐induced ANF‐promoter up‐regulation and increase in cardiomyocyte cell size, and 4) blocking NF‐κB, AKT, or JUN alone or in combination does not sensitize cardiomyocytes to the proapoptotic effects of TNF‐α, in contrast to other cell types, suggesting a cardiac‐specific pathway regulating the anti‐apoptotic events induced by TNF‐α. Altogether, the data presented evidence the role of AKT and JUN in TNF‐αinduced cardiomyocyte hypertrophy and apoptosis.—Condorelli, G., Morisco, C., Latronico, M., Claudio, P. P., Dent, P., Tsichlis, P., Condorelli, G., Frati, G., Drusco, A., Croce, C. M., Napoli, C. TNF‐α signal transduction in rat neonatal cardiac myocytes: definition of pathways generating from the TNF‐α receptor. FASEB J. 16, 1732–1737 (2002)

Insulin Enhances Endothelial α2-Adrenergic Vasorelaxation by a Pertussis Toxin Mechanism

To investigate whether insulin effect on endothelium is related to a specific signal transduction pathway or reflects a more generalized action of the hormone, we studied in aortic rings of Wistar-Kyoto (WKY) rats the effects of the hormone on endothelium-dependent relaxations generated by acetylcholine, adenosine diphosphate, the selective alpha2-adrenergic agonist UK 14,304, and the calcium ionophore ionomycin. The responses were evaluated both in control conditions and after 30 minutes of exposure to three different levels of insulin (30, 100, and 500 microU/mL). Insulin failed to modify the phenylephrine aortic contractions and the relaxations induced by acetylcholine, adenosine diphosphate, and ionomycin. In contrast, both 100 and 500 microU/mL insulin were able to potentiate the UK 14,304-induced vasorelaxation (+96+/-19% and +91+/-12%, respectively). Pertussis toxin, which causes alpha2-adrenergic receptor Gi uncoupling, reduced the alpha2-adrenergic vasorelaxation and prevented the insulin potentiation of the response to UK 14,304. Furthermore, in primary cultured aortic endothelial cells from WKY, we evaluated the conversion of [3H]arginine to [3H]citrulline in response to acetylcholine, ionomycin, and UK 14,304, both in control conditions and during insulin exposure. Again, insulin did not affect basal citrulline production or the increase induced by acetylcholine and ionomycin, whereas it potentiated the response to UK 14,304. Finally, in aortic rings of spontaneously hypertensive rats, insulin treatment (100 and 500 microU/mL) was unable to enhance the alpha2-adrenergic vasodilator response; in vascular endothelial cells from spontaneously hypertensive rats, insulin did not potentiate the increase in citrulline production evoked by UK 14,304. In conclusion, insulin selectively enhances alpha2-adrenergic endothelial vasorelaxation through a pertussis toxin-sensitive mechanism, by potentiating endothelial nitric oxide production. This vasorelaxant mechanism is altered in spontaneously hypertensive rats.

Cross-Talk Between PKA and Akt Protects Endothelial Cells From Apoptosis in the Late Ischemic Preconditioning

Objective—The aim of this study was to explore the molecular mechanisms involved in late preconditioning-induced cell protection in endothelial cells. Methods and Results—Preconditioning (PC) was induced by exposing bovine aortic endothelial cells (BAECs) to 3 cycles of 15 minutes of hypoxia followed by 15 minutes of reoxygenation. A 12-hour period of hypoxia induced cell death in 60% of BAECs (48±5% apoptosis, 12±4% necrosis). Early and late PC decreased hypoxia-induced apoptotic (25±5% and 28±4%, respectively) and necrotic (6±3%, and 8±2%, respectively) cell death. Consistently, hypoxia-induced caspase-3 cleavage was reduced by PC. Pretreatment with H89 (protein kinase A [PKA] inhibitor), LY294002 (phosphatidyl-inositol-3-kinase [PI3K] inhibitor), and N-acetyl-cysteine (antioxidant) abrogated late PC-induced cell protection, whereas inhibition of protein kinase C by Go6983, and of nitric oxide synthesis by L-NAME,1400W and bovine eNOS siRNA did not. In addition, in early and late PC, PKA physically interacted with the phosphorylated form of Akt, suggesting that PKA is required for Akt phosphorylation. Expression of PKA and Akt dominant negative mutants inhibited ischemic late PC-induced protection, indicating that these kinases play a key role in late PC-mediated cell protection. Conclusions—Late ischemic PC protects BAECs against hypoxia through PKA- and PI3K-dependent activation of Akt.

Rest-injected thallium-201 redistribution and resting technetium-99m methoxyisobutylisonitrile uptake in coronary artery disease: relation to the severity of coronary artery stenosis

To compare rest-injected thallium-201 (Tl) redistribution and resting technetium-99m methoxyisobutylisonitrile (99mTc-MIBI) myocardial uptake in chronic coronary artery disease (CAD), 15 patients with angiographically proven CAD and left ventricular (LV) dysfunction (ejection fraction 34%±9%) were studied. All patients underwent rest-redistribution Tl and resting 99mTc-MIBI cardiac imaging. Gated 99mTc-MIBI images were also acquired to assess regional LV wall motion (WM). Myocardial segments (n=225) were divided into three groups on the basis of the degree of coronary artery stenosis: group 1 (total occlusion, n=82), group 2 (50%–99% of stenosis, n=84) and group 3 (<50% of stenosis, n=59). WM was significantly worse in groups 1 and 2 compared to group 3 (P<0.001), but no difference was observed between groups 1 and 2. TI and 99mTc-MIBI uptake were significantly lower in groups 1 and 2 compared to group 3 (P < 0.001), and in group 1 compared to group 2 (P<0.001). When TI and 99mTc-MIBI uptake were directly compared, TI uptake was higher than 99mTc-MIBI uptake in group 1 (P<0.001), while no significant difference was observed in groups 2 and 3. Thus, both rest-injected TI redistribution and resting 99mTc-MIBI uptake reflected the severity of coronary artery stenosis in CAD. However, in myocardial segments with total coronary occlusion T1 uptake was significantly higher than 99mTc-MIBI uptake. Our data suggest that rest-injected Tl redistribution cardiac imaging may identify, more accurately than resting 99mTc-MIBI imaging, the presence of viable myocardium in chronic CAD, particularly when the coronary blood flow is severely impaired.

Characterization of Caveolae from Rat Heart: Localization of Postreceptor Signal Transduction Molecules and Their Rearrangement After Norepinephrine Stimulation

Caveolae are plasma membrane subcompartments that have been implicated in signal transduction. In many cellular systems, caveolae are rich in signal transduction molecules such as G proteins and receptor‐associated tyrosine kinases. An important structural component of the caveolae is caveolin. Recent evidence show that among the caveolin gene family, caveolin‐3 is expressed in skeletal and cardiac muscle and caveolae are present in cardiac myocyte cells. Both the ANP receptor as well as the muscarinic receptor have been localized to the caveolae of cardiac myocytes in culture. These findings prompted us to conduct a further analysis of cardiac caveolae. In order to improve our understanding of the mechanisms of signal transduction regulation in cardiac myocytes, we isolated cardiac caveolae by discontinuous sucrose density gradient centrifugation from rat ventricles and rat neonatal cardiocytes. Our analysis of caveolar content demonstrates that heterotrimeric G proteins, p21ras and receptor‐associated tyrosine kinases are concentrated within these structures. We also show that adrenergic stimulation induces an increase in the amount of diverse α‐ and β‐subunits of G proteins, as well as p21ras, in both in vivo and in vitro experimental settings. Our data show that cardiac caveolae are an important site of signal transduction regulation. This finding suggests a potential role for these structures in physiological and pathological states. J. Cell. Biochem. 77:529–539, 2000.

Distinct Vasodilation, without Reflex Neurohormonal Activation, Induced by Barnidipine in Hypertensive Patients

Barnidipine is a new 1,4-dihydropyridine calcium antagonist with a strong and long-lasting vasodilatory effect. In order to assess the haemodynamic profile of the antihypertensive effect of barnidipine, a randomized, double-blind study of barnidipine vs nitrendipine was performed in 24 patients with mild to moderate essential hypertension. Following an initial 4-week placebo period, patients whose sitting diastolic blood pressure (SiDBP) was between 95 and 114 mm Hg, and whose sitting systolic blood pressure was between 150 and 219 mm Hg, were randomized (2:1 ratio) to receive either barnidipine (10 mg) or nitrendipine (10 mg) once daily, for a 6-week double-blind period. Subsequently, patients with an SiDBP of less than 90 mm Hg continued for a second 6-week period with the same monotherapy, while patients with an SiDBP of 90 mm Hg or above received double the dose of antihypertensive treatment for the next 6 weeks. Two-dimensional M- and B-mode echocardiography with Doppler flowmetry was performed at the end of both the placebo and active treatment phases. Barnidipine and nitrendipine reduced blood pressure by the same degree (barnidipine: from 165 +/- 2/100 +/- 1 to 145 +/- 2/89 +/- 1 mm Hg, p < 0.01; nitrendipine: from 163 +/- 3/100 +/- 2 to 143 +/- 7/90 +/- 3 mm Hg, p < 0.01) as a result of peripheral vasodilation. This was not accompanied by reflex neurohormonal activation. Moreover, only in the group receiving barnidipine was a significant decrease in plasma noradrenaline observed, both when the patients were in the supine position (from 298 +/- 27 to 214 +/- 21 pg/ml, p < 0.05) and when they were upright (from 472 +/- 37 to 348 +/- 38 pg/ml, p < 0.05).