Background
Asymptomatic diabetic patients have a high incidence of clinically unrecognized left ventricular dysfunction with an abnormal cardiac response to exercise. We, therefore, examined ...subclinical defects in the contraction–relaxation cycle and intracellular Ca
2+
regulation in myocardium of asymptomatic type 2 diabetic patients.
Methods
Alterations in the dynamics of the intracellular Ca
2+
transient and contractility were recorded in right atrial myocardium of type 2 diabetic patients and non-diabetic control tissue loaded with fura-2. In order to gain an insight into mechanisms underlying the altered Ca
2+
handling in diabetic myocardium levels of mRNA, protein expression and phosphorylation of key proteins in sarcoplasmic Ca
2+
handling were determined.
Results
In isolated atrial trabeculae of diabetic myocardium the rise of systolic Ca
2+
was significantly prolonged, but relaxation of the Ca
2+
transient was unaltered compared to control tissue. Accordingly, the levels of expression of mRNA and protein of the Ca
2+
release channel (RyR2) of the sarcoplasmic reticulum were reduced by 68 and 22%, respectively. Endogenous phosphorylation of RyR2 by protein kinases C, however, was increased by 31% in diabetic myocardium, as assessed by the back-phosphorylation technique. Levels of expression of SERCA2 and phospholamban were unaltered between both groups.
Conclusions
Intracellular Ca
2+
release is prolonged in non-failing myocardium of type 2 diabetic patients and this may be primarily due to a decreased expression of RyR2. This defective Ca
2+
release may represent an early stage of ventricular dysfunction in type 2 diabetes and would favor the abnormal response to exercise frequently observed in asymptomatic diabetic patients.
Diabetes mellitus counts as a major risk factor for developing atherosclerosis. The activation of protein kinase C (PKC) is commonly known to take a pivotal part in the pathogenesis of ...atherosclerosis, though the influence of specific PKC isozymes remains unclear. There is evidence from large clinical trials suggesting excessive neurohumoral stimulation, amongst other pathways leading to PKC activation, as a central mechanism in the pathogenesis of diabetic heart disease. The present study was therefore designed to determine the role of Gq-protein signalling via Gα11 in diabetes for the expression of PKC isozymes in the coronary vessels.
The role of Gα11 in diabetes was examined in knockout mice with global deletion of Gα11 compared to wildtype controls. An experimental type 1-diabetes was induced in both groups by injection of streptozotocin. Expression and localization of the PKC isozymes α, βII, δ, ε, and ζ was examined by quantitative immunohistochemistry.
8 weeks after induction of diabetes a diminished expression of PKC ε was observed in wildtype animals. This alteration was not seen in Gα11 knockout animals, however, these mice showed a diminished expression of PKCζ. Direct comparison of wildtype and knockout control animals revealed a diminished expression of PKC δ and ε in Gα11 knockout animals.
The present study shows that expression of the nPKCs δ and ε in coronary vessels is under control of the g-protein Gα11. The reduced expression of PKC ζ that we observed in coronary arteries from Gα11-knockout mice compared to wildtype controls upon induction of diabetes could reduce apoptosis and promote plaque stability. These findings suggest a mechanism that may in part underlie the therapeutic benefit of RAS inhibition on cardiovascular endpoints in diabetic patients.
Abstract Aims/hypothesis Pathological cardiac hypertrophy is an early phenotype in both types 1 and 2 diabetes. The primary stimulus for hypertrophic growth in diabetes is yet unknown and may involve ...neurohumoral stimulation of Gq-coupled receptors as well as direct glucose-dependent mechanisms. To discriminate between these hypertrophic stimuli we analyzed hypertrophic signalling pathways in wildtype and Gα11 -knockout mice. Methods Experimental diabetes was induced in wildtype and knockout mice by intraperitoneal injection of streptozotocin. 8 weeks after induction of diabetes myocardial function and structure was assessed by echocardiography before sacrifice. To identify prohypertrophic signalling pathways expression and translocation of protein kinase C isoforms α, βII , δ, ε and ζ were analyzed by immunohistochemical staining and immunoblot analysis after tissue fractionation. Changes in calcineurin signalling were identified by immunoblot analysis and functional assays. Expression levels of transcription factors GATA4 and NF-κB were quantified by real-time RT-PCR. Results Diabetic wildtype mice developed myocardial hypertrophy with preserved cardiac function. Calcineurin signalling was not different between the two groups. However, diabetic wildtype mice showed increased protein levels of PKC-α and PKC-ζ, translocation of PKC-α, -δ and -ε to cellular membranes and higher levels of NF-κB expression. In contrast, diabetic Gα11 -knockout mice showed no altered phenotype and no changes in NF-κB or PKC expression, although translocation of PKC-ε occurred as in wildtypes. Conclusions Gα11 is essential for the development of cardiac hypertrophy in type 1-diabetes. Stimulation of hypertrophic signalling through PKC-α, PKC-δ, PKC-ζ, and NF-κB appears to be receptor-dependent, whereas PKC-ε is activated by hyperglycemia, independent of Gα11.
Neurohumoral stimulation of Gq-coupled receptors has been proposed as a central mechanism in the pathogenesis of diabetic heart disease. The resulting contractile dysfunction is closely related to ...abnormal intracellular Ca(2+) handling with functional defects of the sarcoplasmic reticulum (SR). The present study was therefore designed to determine the role of G(q)-protein signaling via G(alpha)(11) and G(alpha)(q) in diabetes for the induction of functional and structural changes in the Ca(2+) release complex of the SR. An experimental type 1-diabetes was induced in wild type, G(alpha)(11) knockout, and G(alpha)(11/q)-knockout mice by injection of streptozotocin. Cardiac morphology and function was assessed in vivo by echocardiography. SR Ca(2+) leak was tested in vitro based on a (45)Ca(2+) assay and protein densities as well as gene expression of ryanodine receptor (RyR2), FKBP12.6, sorcin, and annexin A7 were analyzed by immunoblot and RT-PCR. In wild type animals 8 weeks of diabetes resulted in cardiac hypertrophy and SR Ca(2+) leak was increased. In addition, diabetic wild type animals showed reduced protein levels of FKBP12.6 and annexin A7. In G(alpha)(11)- and G(alpha)(11/q)-knockout animals, however, SR Ca(2+) release and cardiac phenotype remained unchanged upon induction of diabetes. Densities of the proteins that we presently analyzed were also unaltered in G(alpha)(11)-knockout mice. G(alpha)(11/q)-knockout animals even showed increased expression of sorcin and annexin A7. Thus, based on the present study we suggest a signaling pathway via the G(q)-proteins, G(alpha)(11) and G(alpha)(q), that could link increased neurohumoral stimulation in diabetes with defective RyR2 channel function by regulating protein expression of FKBP12.6, annexin A7, and sorcin.
Neurohumoral stimulation of Gq-coupled receptors has been proposed as a central mechanism in the pathogenesis of diabetic heart disease. The resulting contractile dysfunction is closely related to ...abnormal intracellular Ca²⁺ handling with functional defects of the sarcoplasmic reticulum (SR). The present study was therefore designed to determine the role of Gq-protein signaling via Gα₁₁ and Gαq in diabetes for the induction of functional and structural changes in the Ca²⁺ release complex of the SR. An experimental type 1-diabetes was induced in wild type, Gα₁₁ knockout, and Gα₁₁/q-knockout mice by injection of streptozotocin. Cardiac morphology and function was assessed in vivo by echocardiography. SR Ca²⁺ leak was tested in vitro based on a ⁴⁵Ca²⁺ assay and protein densities as well as gene expression of ryanodine receptor (RyR2), FKBP12.6, sorcin, and annexin A7 were analyzed by immunoblot and RT-PCR. In wild type animals 8 weeks of diabetes resulted in cardiac hypertrophy and SR Ca²⁺ leak was increased. In addition, diabetic wild type animals showed reduced protein levels of FKBP12.6 and annexin A7. In Gα₁₁- and Gα₁₁/q-knockout animals, however, SR Ca²⁺ release and cardiac phenotype remained unchanged upon induction of diabetes. Densities of the proteins that we presently analyzed were also unaltered in Gα₁₁-knockout mice. Gα₁₁/q-knockout animals even showed increased expression of sorcin and annexin A7. Thus, based on the present study we suggest a signaling pathway via the Gq-proteins, Gα₁₁ and Gαq, that could link increased neurohumoral stimulation in diabetes with defective RyR2 channel function by regulating protein expression of FKBP12.6, annexin A7, and sorcin.
Abstract Background: Diabetes mellitus counts as a major risk factor for developing atherosclerosis. The activation of protein kinase C (PKC) is commonly known to take a pivotal part in the ...pathogenesis of atherosclerosis, though the influence of specific PKC isozymes remains unclear. There is evidence from large clinical trials suggesting excessive neurohumoral stimulation, amongst other pathways leading to PKC activation, as a central mechanism in the pathogenesis of diabetic heart disease. The present study was therefore designed to determine the role of Gq -protein signalling via Gα11 in diabetes for the expression of PKC isozymes in the coronary vessels. Methods: The role of Gα11 in diabetes was examined in knockout mice with global deletion of Gα11 compared to wildtype controls. An experimental type 1-diabetes was induced in both groups by injection of streptozotocin. Expression and localization of the PKC isozymes α, βII, δ, straight epsilon, and ζ was examined by quantitative immunohistochemistry. Results: 8 weeks after induction of diabetes a diminished expression of PKC straight epsilon was observed in wildtype animals. This alteration was not seen in Gα11 knockout animals, however, these mice showed a diminished expression of PKCζ. Direct comparison of wildtype and knockout control animals revealed a diminished expression of PKC δ and straight epsilon in Gα11 knockout animals. Conclusion: The present study shows that expression of the nPKCs δ and straight epsilon in coronary vessels is under control of the g-protein Gα11 . The reduced expression of PKC ζ that we observed in coronary arteries from Gα11 -knockout mice compared to wildtype controls upon induction of diabetes could reduce apoptosis and promote plaque stability. These findings suggest a mechanism that may in part underlie the therapeutic benefit of RAS inhibition on cardiovascular endpoints in diabetic patients.
Zusammenfassung
Studienergebnisse der Vergangenheit stellen den therapeutischen Nutzen der Lungenmetastasenchirurgie für Patienten mit pulmonal metastasiertem malignem Melanom infrage. Eine ...systematische Literaturrecherche und eine Analyse der Artikel, die ab dem 01.01.2000 veröffentlicht worden sind, sollen den Nutzen überprüfen und therapeutische Alternativen ermitteln. Nach der Metastasektomie erreichten die Patienten ein medianes Überleben von 17 bis 40 Monaten und 5-Jahres-Überlebensraten von 18–39,4%. Bis zu 30% der Patienten erlitten intrathorakale Rezidive, die in einigen Fällen durch Reoperationen erfolgreich therapiert worden sind. Verschiedene monoklonale Antikörper stehen als Behandlungsalternativen zur Verfügung und ermöglichen derzeit ein medianes Überleben von bis zu 17 Monaten. Die Metastasektomie ist für selektionierte Patienten die Therapie der Wahl, ihr Nutzen muss zukünftig jedoch im Vergleich zu den Pharmaka der aktuellen Generation erneut validiert werden.
Results of previous studies question the benefits of pulmonary surgery in patients with pulmonary metastases from malignant melanoma. A systematic literature search and analysis of articles published ...since 1 January 2000 was carried out to investigate the advantages of metastasectomy and alternative forms of therapy. Patients reached a median survival time of 17-40 months and 5-year survival rates between 18% and 39.4% after metastasectomy. Intrathoracic recurrence occurred in 30 % of patients but could be successfully treated with re-operations in some cases. Various monoclonal antibodies are currently available and achieve a median survival time of up to 17 months. Pulmonary metastasectomy is the treatment of choice in selected patients; however, in the future the benefits should be revalidated in comparison with pharmaceuticals of the current generation.
Neurohumoral stimulation of Gq-coupled receptors has been proposed as a central mechanism in the pathogenesis of diabetic heart disease. The resulting contractile dysfunction is closely related to ...abnormal intracellular Ca.sup.2+ handling with functional defects of the sarcoplasmic reticulum (SR). The present study was therefore designed to determine the role of G.sub.q-protein signaling via Galpha.sub.11 and Gaq in diabetes for the induction of functional and structural changes in the Ca.sup.2+ release complex of the SR. An experimental type 1-diabetes was induced in wild type, Galpha.sub.11 knockout, and Galpha.sub.11/q- knockout mice by injection of streptozotocin. Cardiac morphology and function was assessed in vivo by echocardiography. SR Ca.sup.2+ leak was tested in vitro based on a sup.45Ca.sup.2+ assay and protein densities as well as gene expression of ryanodine receptor (RyR2), FKBP12.6, sorcin, and annexin A7 were analyzed by immunoblot and RT-PCR. In wild type animals 8 weeks of diabetes resulted in cardiac hypertrophy and SR Ca.sup.2+ leak was increased. In addition, diabetic wild type animals showed reduced protein levels of FKBP12.6 and annexin A7. In Galpha.sub.11- and Galpha.sub.11/q-knockout animals, however, SR Ca.sup.2+ release and cardiac phenotype remained unchanged upon induction of diabetes. Densities of the proteins that we presently analyzed were also unaltered in Galpha.sub.11-knockout mice. Galpha.sub.11/q-knockout animals even showed increased expression of sorcin and annexin A7. Thus, based on the present study we suggest a signaling pathway via the G.sub.q-proteins, Galpha.sub.11 and Galpha.sub.q, that could link increased neurohumoral stimulation in diabetes with defective RyR2 channel function by regulating protein expression of FKBP12.6, annexin A7, and sorcin. Keywords Ryanodine receptor * Type 1-diabetes * Knockout mice * Sorcin * Annexin A7
The TOR and Insulin/IGF signalling (IIS) network controls growth, metabolism and ageing. Although reducing TOR or insulin signalling can be beneficial for ageing, it can be detrimental for wound ...healing, but the reasons for this difference are unknown. Here we show that IIS is activated in the cells surrounding an epidermal wound in Drosophila melanogaster larvae, resulting in PI3K activation and redistribution of the transcription factor FOXO. Insulin and TOR signalling are independently necessary for normal wound healing, with FOXO and S6K as their respective effectors. IIS is specifically required in cells surrounding the wound, and the effect is independent of glycogen metabolism. Insulin signalling is needed for the efficient assembly of an actomyosin cable around the wound, and constitutively active myosin II regulatory light chain suppresses the effects of reduced IIS. These findings may have implications for the role of insulin signalling and FOXO activation in diabetic wound healing.
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