Mathias Konstandin

MYOCARDIAL AKT: THE OMNIPRESENT NEXUS

One of the greatest examples of integrated signal transduction is revealed by examination of effects mediated by AKT kinase in myocardial biology. Positioned at the intersection of multiple afferent and efferent signals, AKT exemplifies a molecular sensing node that coordinates dynamic responses of the cell in literally every aspect of biological responses. The balanced and nuanced nature of homeostatic signaling is particularly essential within the myocardial context, where regulation of survival, energy production, contractility, and response to pathological stress all flow through the nexus of AKT activation or repression. Equally important, the loss of regulated AKT activity is primarily the cause or consequence of pathological conditions leading to remodeling of the heart and eventual decompensation. This review presents an overview compendium of the complex world of myocardial AKT biology gleaned from more than a decade of research. Summarization of the widespread influence that AKT exerts upon myocardial responses leaves no doubt that the participation of AKT in molecular signaling will need to be reckoned with as a seemingly omnipresent regulator of myocardial molecular biological responses.

The serine phosphatases PP1 and PP2A associate with and activate the actin‐binding protein cofilin in human T lymphocytes

Cofilin, an actin‐depolymerizing protein, is essential for the functional dynamics of the actin cytoskeleton and for cell viability. In unstimulated human peripheral blood T lymphocytes cofilin is phosphorylated and localized in the cytoplasm. Following co‐stimulation through accessory receptors (e.g. CD2 or CD28) – however, not following TCR/CD3 stimulation alone – cofilin undergoes dephosphorylation. The subcellular localization as well as the actin‐binding activity of cofilin are regulated by the phosphorylation state of serine‐3. Thus, only the dephosphorylated form of cofilin associates with the actin cytoskeleton and possesses the capability to translocate into the nucleus. Recently, LIM‐kinase 1 was shown to inactivate cofilin through phosphorylation. Here, we have identified the functional counterparts of LIM‐kinase 1: the serine/threonine phosphatases of type 1 and type 2A not only associate with cofilin but also dephosphorylate this 19‐kDa protein and thereby mediate cofilin activation. In malignant T lymphoma cells, activation of these phosphatases occurs spontaneously, independent of external stimuli. In untransformed human peripheral blood T lymphocytes, these phosphatases function through a cyclosporin A/FK506‐resistant co‐stimulatory signaling pathway which is common for the accessory receptors CD2 and CD28. This co‐stimulatory signaling pathway is also not affected by a series of other clinically established immunosuppressive drugs (i.e. rapamycin, dexamethasone, leflunomide or mycophenolic acid).

High affinity interaction of integrin α4β1 (VLA-4) and vascular cell adhesion molecule 1 (VCAM-1) enhances migration of human melanoma cells across activated endothelial cell layers

The capacity of tumor cells to form metastatic foci correlates with their ability to interact with and migrate through endothelial cell layers. This process involves multiple adhesive interactions between tumor cells and the endothelium. Only little is known about the molecular nature of these interactions during extravasation of tumor cells. In human melanoma cells, the integrin αvβ3 is involved in transendothelial migration and its expression correlates with metastasis. However, many human melanoma cells do not express β3 integrins. Therefore, it remained unclear how these cells undergo transendothelial migration. In this study we show that human melanoma cells with different metastatic potency, which do not express β2 or β3 integrins, express the VCAM‐1 receptor α4β1. VCAM‐1 is up‐regulated on activated endothelial cells and is known to promote transendothelial migration of leukocytes. Interestingly, despite comparable cell surface levels of α4β1, only the highly metastatic melanoma cell lines MV3 and BLM, but not the low metastatic cell lines IF6 and 530, bind VCAM‐1 with high affinity without further stimulation, and are therefore able to adhere to and migrate on isolated VCAM‐1. Moreover, we demonstrate that function‐blocking antibodies against the integrin α4β1, as well as siRNA‐mediated knock‐down of the α4 subunit in these highly metastatic human melanoma cells reduce their transendothelial migration. These data imply that only high affinity interactions between the integrin α4β1 on melanoma cells and VCAM‐1 on activated endothelial cells may enhance the metastatic capacity of human β2/β3‐negative melanoma cells. J. Cell. Physiol. 212: 368–374, 2007.

Pharmacodynamic Monitoring of Cyclosporine A in Renal Allograft Recipients Shows a Quantitative Relationship Between Immunosuppression and the Occurrence of Recurrent Infections and Malignancies

Background. At present it is unclear which dose and consecutive blood levels of cyclosporine A (CsA) are optimal with respect to immunosuppressive efficacy and drug specific side effects at the level of individual patients. Several pharmacodynamic measures of CsA effects have been proposed, but have not become clinical routine yet. Besides the lack of practicability, the biological relevance of these assays has not been determined so far. Methods. Residual expression of nuclear factor of activated T-cells (NFAT)-regulated genes two hours after drug intake was used as molecular pharmacodynamic marker to assess CsA effects on lymphocytes and correlated with the frequency of recurrent infections and malignancies in patients with five or more years of follow-up posttransplantation. Results. Recurrent infectious complications were observed in 44% and malignancies in 20% of the 133 patients studied. Patients with a strong suppression of NFAT-regulated genes by CsA—as judged by a residual level of transcription of less than 15% after drug intake—develop more frequent infections (53% vs. 29%; P=0.005) and malignancies (22% vs. 4%; P=0.002). The lack of correlation between the incidence of these complications and CsA blood concentration might point to the interindividual differences in the sensitivity towards calcineurin inhibition. Conclusion. The data presented here reveal a clear relation between the frequency of infectious and malignant complications and the degree of suppression of NFAT-regulated genes by CsA in transplanted patients. Therefore, pharmacodynamic monitoring of CsA efficacy in transplanted patients might be a useful tool to adjust immunosuppressive therapy in individual patients.

Mitochondrial translocation of Nur77 mediates cardiomyocyte apoptosis

AIMS The cascade of events leading to compromised mitochondrial integrity in response to stress is mediated by various combinatorial interactions of pro- and anti-apoptotic molecules. Nur77, an immediate early gene that encodes a nuclear orphan receptor, translocates from the nucleus to mitochondria to induce cytochrome c release and apoptosis in cancer cells in response to various pro-apoptotic treatments. However, the role of Nur77 in the cardiac setting is still unclear. The objective of this study is to determine the physiological relevance and pathophysiological importance of Nur77 in cardiomyocytes. METHODS AND RESULTS Myocardial Nur77 is upregulated following cardiomyopathic injury and, while expressed in the postnatal myocardium, declines in level within weeks after birth. Nur77 is localized predominantly in cardiomyocyte nuclei under normal conditions where it is not apoptotic, but translocates to mitochondria in response to oxidative stress both in vitro and in vivo. Mitochondrial localization of Nur77 induces cytochrome c release and typical morphological features of apoptosis, including chromatin condensation and DNA fragmentation. Knockdown of Nur77 rescued hydrogen peroxide-induced cardiomyocyte apoptosis. CONCLUSION Translocation of Nur77 from the nucleus to the mitochondria in cardiomyocytes results in the loss of mitochondrial integrity and subsequent apoptosis in response to ischaemia/reperfusion injury. Our findings identify Nur77 as a novel mediator of cardiomyocyte apoptosis and warrants further investigation of mitochondrial Nur77 translocation as a mechanism to control cell death in the treatment of ischaemic heart diseases.

Sustained LFA‐1 cluster formation in the immune synapse requires the combined activities of L‐plastin and calmodulin

Formation of immune synapses (IS) between T cells and APC requires multiple rearrangements in the actin cytoskeleton and selective receptor accumulation in supramolecular activation clusters (SMAC). The inner cluster (central SMAC) contains the TCR/CD3 complex. The outer cluster (peripheral SMAC) contains the integrin LFA‐1 and Talin. Molecular mechanisms selectively stabilizing receptors in the IS remained largely unknown. Here, we demonstrate that sustained LFA‐1 clustering in the IS is a consequence of the combined activities of the actin‐bundling protein L‐plastin (LPL) and calmodulin. Thus, upon antigen‐recognition of T cells, LPL accumulated predominantly in the peripheral SMAC. siRNA‐mediated knock‐down of LPL led to a failure of LFA‐1 and Talin redistribution – however, not TCR/CD3 relocalization – into the IS. As a result of this LPL knock‐down, the T‐cell/APC interface became smaller over time and T‐cell proliferation was inhibited. Importantly, binding of calmodulin to LPL was required for the maintenance of LPL in the IS and consequently inhibition of calmodulin also prevented stable accumulation of LFA‐1 and Talin, but not CD3, in the IS.

Fibronectin Is Essential for Reparative Cardiac Progenitor Cell Response After Myocardial Infarction

Rationale: Adoptive transfer of cardiac progenitor cells (CPCs) has entered clinical application, despite limited mechanistic understanding of the endogenous response after myocardial infarction (MI). Extracellular matrix undergoes dramatic changes after MI and therefore might be linked to CPC-mediated repair. Objective: To demonstrate the significance of fibronectin (Fn), a component of the extracellular matrix, for induction of the endogenous CPC response to MI. Methods and Results: This report shows that presence of CPCs correlates with the expression of Fn during cardiac development and after MI. In vivo, genetic conditional ablation of Fn blunts CPC response measured 7 days after MI through reduced proliferation and diminished survival. Attenuated vasculogenesis and cardiogenesis during recovery were evident at the end of a 12-week follow-up period. Impaired CPC-dependent reparative remodeling ultimately leads to continuous decline of cardiac function in Fn knockout animals. In vitro, Fn protects and induces proliferation of CPCs via &bgr;1-integrin-focal adhesion kinase-signal transducer and activator of transcription 3-Pim1 independent of Akt. Conclusions: Fn is essential for endogenous CPC expansion and repair required for stabilization of cardiac function after MI.

Pim-1 preserves mitochondrial morphology by inhibiting dynamin-related protein 1 translocation.

Mitochondrial morphological dynamics affect the outcome of ischemic heart damage and pathogenesis. Recently, mitochondrial fission protein dynamin-related protein 1 (Drp1) has been identified as a mediator of mitochondrial morphological changes and cell death during cardiac ischemic injury. In this study, we report a unique relationship between Pim-1 activity and Drp1 regulation of mitochondrial morphology in cardiomyocytes challenged by ischemic stress. Transgenic hearts overexpressing cardiac Pim-1 display reduction of total Drp1 protein levels, increased phosphorylation of Drp1-S637, and inhibition of Drp1 localization to the mitochondria. Consistent with these findings, adenoviral-induced Pim-1 neonatal rat cardiomyocytes (NRCMs) retain a reticular mitochondrial phenotype after simulated ischemia (sI) and decreased Drp1 mitochondrial sequestration. Interestingly, adenovirus Pim-dominant negative NRCMs show increased expression of Bcl-2 homology 3 (BH3)-only protein p53 up-regulated modulator of apoptosis (PUMA), which has been previously shown to induce Drp1 accumulation at mitochondria and increase sensitivity to apoptotic stimuli. Overexpression of the p53 up-regulated modulator of apoptosis–dominant negative adenovirus attenuates localization of Drp1 to mitochondria in adenovirus Pim-dominant negative NRCMs promotes reticular mitochondrial morphology and inhibits cell death during sI. Therefore, Pim-1 activity prevents Drp1 compartmentalization to the mitochondria and preserves reticular mitochondrial morphology in response to sI.

Mechanistic Target of Rapamycin Complex 2 Protects the Heart From Ischemic Damage

Background— The mechanistic target of rapamycin (mTOR) comprises 2 structurally distinct multiprotein complexes, mTOR complexes 1 and 2 (mTORC1 and mTORC2). Deregulation of mTOR signaling occurs during and contributes to the severity of myocardial damage from ischemic heart disease. However, the relative roles of mTORC1 versus mTORC2 in the pathogenesis of ischemic damage are unknown. Methods and Results— Combined pharmacological and molecular approaches were used to alter the balance of mTORC1 and mTORC2 signaling in cultured cardiac myocytes and in mouse hearts subjected to conditions that mimic ischemic heart disease. The importance of mTOR signaling in cardiac protection was demonstrated by pharmacological inhibition of both mTORC1 and mTORC2 with Torin1, which led to increased cardiomyocyte apoptosis and tissue damage after myocardial infarction. Predominant mTORC1 signaling mediated by suppression of mTORC2 with Rictor similarly increased cardiomyocyte apoptosis and tissue damage after myocardial infarction. In comparison, preferentially shifting toward mTORC2 signaling by inhibition of mTORC1 with PRAS40 led to decreased cardiomyocyte apoptosis and tissue damage after myocardial infarction. Conclusions— These results suggest that selectively increasing mTORC2 while concurrently inhibiting mTORC1 signaling is a novel therapeutic approach for the treatment of ischemic heart disease.Background —The mechanistic target of rapamycin (mTOR) is comprised of two structurally distinct multiprotein complexes, mTOR complexes 1 and 2 (mTORC1 and 2). Deregulation of mTOR signaling occurs during and contributes to the severity of myocardial damage from ischemic heart disease. However, the relative roles of mTORC1 versus mTORC2 in the pathogenesis of ischemic damage are unknown. Methods and Results —Combined pharmacological and molecular approaches were used to alter the balance of mTORC1 and mTORC2 signaling in cultured cardiac myocytes and in mouse hearts subjected to conditions that mimic ischemic heart disease. The importance of mTOR signaling in cardiac protection was demonstrated by pharmacological inhibition of both mTORC1 and mTORC2 with Torin1, which led to increased cardiomyocyte apoptosis and tissue damage after myocardial infarction (MI). Predominant mTORC1 signaling mediated by suppression of mTORC2 with Rictor similarly increased cardiomyocyte apoptosis and tissue damage after MI. In comparison, preferentially shifting toward mTORC2 signaling by inhibition of mTORC1 with PRAS40 led to decreased cardiomyocyte apoptosis and tissue damage after MI. Conclusions —These results suggest that selectively increasing mTORC2 while concurrently inhibting of mTORC1 signaling is a novel therapeutic approach for the treatment of ischemic heart disease.

Costimulation induced phosphorylation of L-plastin facilitates surface transport of the T cell activation molecules CD69 and CD25.

Rearrangements in the actin cytoskeleton play a pivotal role for costimulation‐induced formation of the immunological synapse and T cell activation. Yet, little is known about the actin‐binding proteins that link costimulation to rearrangements in the actin cytoskeleton. Here we demonstrate that phosphorylation of the actin bundling protein L‐plastin in response to costimulation through TCR/CD3 plus CD2 or CD28, respectively, is important for the activation of human peripheral blood T lymphocytes (PBT). Mass spectrometry and site‐directed mutagenesis revealed that Ser5 represents the only phospho‐acceptor site of L‐plastin in PBT. Wild‐type L‐plastin (wt‐LPL) and a non‐phosphorylatable 5A‐L‐plastin (5A‐LPL) equally relocalized to the immunological synapse between PBT and APC. Yet importantly, cells expressing 5A‐LPL showed a significantly lower expression of the T cell activation molecules CD25 and CD69 on the cell surface than cells expressing wt‐LPL. This effect is due to a failure in the transport of CD25 and CD69 to the cell surface since the total amount of these proteins within the cells remained unchanged. In conclusion, phosphorylation of the actin bundling protein L‐plastin represents a so‐far‐unknown mechanism by which costimulation controls the transport of activation receptors to the T cell surface.