Cantharidin increases the force of contraction and protein phosphorylation in isolated human atria.

Cantharidin, an inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), is known to increase the force of contraction and shorten the time to relaxation in human ventricular preparations. We hypothesized that cantharidin has similar positive inotropic effects in human right atrial appendage (RAA) preparations. RAA were obtained during bypass surgery performed on human patients. These trabeculae were mounted in organ baths and electrically stimulated at 1 Hz. For comparison, we studied isolated electrically stimulated left atrial (LA) preparations and isolated spontaneously beating right atrial (RA) preparations from wild-type mice. Cumulatively applied (starting at 10 to 30 µM), cantharidin exerted a positive concentration-dependent inotropic effect that plateaued at 300 µM in the RAA, LA, and RA preparations. This positive inotropic effect was accompanied by a shortening of the time to relaxation in human atrial preparations (HAPs). Notably, cantharidin did not alter the beating rate in the RA preparations. Furthermore, cantharidin (100 µM) increased the phosphorylation state of phospholamban and the inhibitory subunit of troponin I in RAA preparations, which may account for the faster relaxation observed. The generated data indicate that PP1 and/or PP2A play a functional role in human atrial contractility.

Human histamine H2 receptors can initiate cardiac arrhythmias in a transgenic mouse.

Histamine is known to lead to arrhythmias in the human heart. A mouse model to mimic these effects has hitherto not been available but might be useful to study the mechanism(s) of H2-histamine receptor-induced arrhythmias and may support the search for new antiarrhythmic drugs. In order to establish such a model in mice, we studied here the incidence of cardiac arrhythmias under basal and under stimulated conditions in atrial and ventricular preparations from mice that overexpressed the human H2-histamine receptors in a cardiac-specific way (H2-TG) in comparison with their wild-type (WT) littermate controls. We had shown before that histamine exerted concentration and time-dependent positive inotropic and positive chronotropic effects only in cardiac preparations from H2-TG and not from WT. We noted under basal conditions (no drug addition) that right atrial preparations from H2-TG exhibited more spontaneous arrhythmias than right atrial preparations from WT. These arrhythmias in H2-TG could be blocked by the H2-histamine receptor antagonist cimetidine. In a similar fashion, histamine and dimaprit (an agonist at H2 and not H1-histamine receptors) more often induced arrhythmias in right atrial preparations from H2-TG than from WT. To understand better the signal transduction mechanism(s) involved in these arrhythmias, we studied partially depolarized left atrial preparations. In these preparations, a positive inotropic effect of histamine was still present in the additional presence of 44 mM potassium ions (used to block sodium channels) in H2-TG but not WT and this positive inotropic effect could be blocked by cimetidine and this is consistent with the involvement of calcium ion channels in the contractile and thus might mediate also the arrhythmogenic effects of histamine in H2-TG. However, compounds reported to release histamine from cells and thereby leading to arrhythmias in humans, namely morphine, ketamine, and fentanyl, failed to induce a more pronounced positive inotropic effect in atrial preparations from H2-TG compared to WT, arguing against an involvement of histamine release in their proarrhythmic side effects in patients. Measuring left ventricular contractility in isolated retrogradely perfused hearts (Langendorff mode), we detected under basal conditions (no drug application) more spontaneous arrhythmias in hearts from H2-TG than from WT. In summary, we noted that overexpression of human H2-histamine receptors in a novel transgenic animal model can lead to arrhythmias. We suggest that this model might be useful to understand the mechanism(s) of histamine-induced cardiac arrhythmias in humans better in a molecular way and may be of value to screen novel antiarrhythmic drugs.

Role of Cardiac A2A Receptors Under Normal and Pathophysiological Conditions.

This review presents an overview of cardiac A2A-adenosine receptors The localization of A2A-AR in the various cell types that encompass the heart and the role they play in force regulation in various mammalian species are depicted. The putative signal transduction systems of A2A-AR in cells in the living heart, as well as the known interactions of A2A-AR with membrane-bound receptors, will be addressed. The possible role that the receptors play in some relevant cardiac pathologies, such as persistent or transient ischemia, hypoxia, sepsis, hypertension, cardiac hypertrophy, and arrhythmias, will be reviewed. Moreover, the cardiac utility of A2A-AR as therapeutic targets for agonistic and antagonistic drugs will be discussed. Gaps in our knowledge about the cardiac function of A2A-AR and future research needs will be identified and formulated.

On inotropic effects of UTP in the human heart.

Uridine 5'-triphosphate (UTP) exerts a positive inotropic effect (PIE) in isolated electrically driven isolated right atrial trabeculae carneae from patients undergoing heart surgery. This review discusses some aspects of the current knowledge on the putative receptor(s) involved and the potential biochemical transduction steps leading to the PIE.

Initial Characterization of Transgenic Mice Overexpressing Human Histamine H2 Receptors.

In an integrative approach, we studied the role of histamine H2 receptors in the mouse heart. We noted that histamine, added cumulatively to the organ bath, failed to affect the force of contraction in left atrial preparations and did not change spontaneous heart rate in right atrial preparations from wild-type mice. By contrast, in the same preparations from mice that overexpressed the human H2 receptor in a cardiac-specific way, histamine exerted concentration- and time-dependent positive inotropic and positive chronotropic effects. Messenger RNA of the human H2 receptor was only detected in transgenic mice. Likewise, immunohistology and autoradiography only gave signals in transgenic but not in wild-type cardiac preparations. Similarly, a positive inotropic and positive chronotropic effect was observed with histamine in echocardiography of living transgenic mice and isolated perfused hearts (Langendorff preparation). Phosphorylation of phospholamban was increased in atrial and ventricular preparations from transgenic mice, but not in wild-type animals. The effects of histamine were mimicked by dimaprit and amthamine and antagonized by cimetidine. In summary, we generated a new model to study the physiologic and pathophysiologic cardiac role of the human H2 receptor.

Evidence for a functional role of calsequestrin 2 in mouse atrium.

AIM: Several genetically modified mice models were studied so far to investigate the role of cardiac calsequestrin (CSQ2) for the contractile function of the ventricle and for the occurrence of ventricular tachycardia. Using a CSQ2 knockout mouse, we wanted to study also the atrial function of CSQ2. METHODS: The influence of CSQ2 on atrial function and, for comparison, ventricular function was studied in isolated cardiac preparations and by echocardiography as well as electrocardiography in mice with deletion of CSQ2. RESULTS: Using deletion of exon 1, we have successfully generated a constitutive knockout mouse of the calsequestrin 2 gene (CSQ2-/- ). CSQ2 protein was absent in the heart (atrium, ventricle), but also in oesophagus and skeletal muscle of homozygous knockout mice. In 6-month-old CSQ2-/- mice, relative left atrial weight was increased, whereas relative heart weight was unchanged. The staircase phenomena in paced left atrial preparations on force of contraction and the post-rest potentiation were different between wild type and CSQ2-/- indicative for a decreased sarcoplasmic Ca2+ load and supporting an important role of CSQ2 also in the atrium. The incidence of arrhythmias was increased in CSQ2-/- . In 2-year-old CSQ2-/- mice, cardiac hypertrophy and heart failure were noted possibly as a result of chronically increased cytosolic Ca2+ levels. CONCLUSION: These data suggest a functional role of CSQ2 not only in the ventricle but also in the atrium of mammalian hearts. Loss of CSQ2 function can cause not only arrhythmias, but also cardiac hypertrophy and heart failure.

Overexpression of protein phosphatase 2A in a murine model of chronic myocardial infarction leads to increased adverse remodeling but restores the regulation of ß-catenin by glycogen synthase kinase 3ß.

BACKGROUND/OBJECTIVES: Increased activity of cardiac protein phosphatases is an important feature in human heart failure. Several different protein phosphatases (PP) are involved in the regulation of excitation-contraction-coupling of the myocardium. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase consisting of a dimeric core enzyme and tissue-specific subunits. In this study we used transgenic mice overexpressing PP2A to further investigate the role of PP2A in cardiac remodeling after myocardial infarction. METHODS AND RESULTS: Adult male CD-1 mice overexpressing the catalytic subunit α of PP2A (αMHC-PP2A; TG) underwent chronic LAD-ligation or sham surgery, respectively; wildtype littermates (WT) were used as controls. Cardiac function was determined by echocardiography before and 28 days after LAD-ligation. 28 days after MI, the animals were sacrificed and cardiac remodeling was analyzed in histological sections and by Western blots. PP2A overexpression leads to dilated cardiomyopathy in mice, and increased cardiomyocyte hypertrophy and fibrosis of the remote myocardium can be seen after myocardial infarction. However, we found an improved survival of TG in the subacute phase after MI in comparison to WT. On the molecular level, TG shows reduced expression of SERCA and CaMKII alpha both under basal condition as well 28 days after MI. Additionally, the regulation of the Akt/GSK3ß/ß-catenin pathway is severely disturbed in TG at baseline where a significant activation of Akt is found that coincides with the typical phosphorylation of GSK3ß. However, this does not lead to the accumulation of ß-catenin - on the contrary: phosphorylation-induced degradation of ß-catenin is significantly enhanced. CONCLUSION: Transgenic overexpression of myocardial PP2A causes adverse remodeling which coincides with a disruption of the classical Akt/GSK3/ß-catenin pathway under baseline conditions that is restored to normal values in chronic myocardial infarction. Even so overall survival of TG after myocardial infarction was not constrained and survival after day 2 post MI was improved.

Tumor necrosis factor-alpha at acute myocardial infarction in rats and effects on cardiac fibroblasts.

Tumor necrosis factor-alpha (TNF-alpha) biosynthesis by the myocardium in response to several diseases has not been solely associated with activation of the immune system but may also serve as a stress response in the context of neurohumoral gene activation. In this regard, beneficial as well as adverse effects of the cytokine on injured myocardium have been reported. TNF-alpha has been suggested to modulate myocyte and fibroblast cell growth and function. Now, in a rat model of acute myocardial infarction TNF-alpha expression and effects on cardiac fibroblast were determined. TNF-alpha was detected in rat hearts with acute myocardial infarction, parallel to the presence of proliferating fibroblasts, at the border zone of the infarct region, to a lesser degree in the infarct zone and was still present in the surviving myocardium. Similarly, the TNF-alpha mRNA level was, compared to sham-operated heart, higher in the infarct area. In the remote myocardium, a trend to an elevated TNF-alpha mRNA level was observed. TNF-alpha stimulated proliferation and expression of fibronectin in fibroblasts isolated from the infarct, non-infarct-region and sham-operated hearts. Angiotensin II is mitogenic for fibroblasts post-myocardial infarction and effects might be mediated indirectly by TNF-alpha. Addition of a neutralising anti-TNF-alpha antibody inhibited angiotensin II stimulated proliferation of fibroblasts only from the infarcted myocardium. The regional differences in TNF-alpha protein and mRNA levels, parallel to proliferating fibroblasts and proliferative effects may foster the reparative, reactive and adverse post-infarct remodeling of the heart.

Immunochemical characterization of the gap junction protein connexin45 in mouse kidney and transfected human HeLa cells.

Antibodies to the gap junction protein connexin45 (Cx45) were obtained by immunizing rabbits with fusion protein consisting of glutathione S-transferase and 138 carboxy-terminal amino acids of mouse Cx45. As shown by immunoblotting and immunofluorescence, the affinity-purified antibodies recognized Cx45 protein in transfected human HeLa cells as well as in the kidney-derived human and hamster cell lines 293 and BHK21, respectively. In Cx45-transfected HeLa cells, this protein is phosphorylated as demonstrated by immunoprecipitation after metabolic labeling. The phosphate label could be removed by treatment with alkaline phosphatase. A weak phosphorylation of Cx45 protein was also detected in the cell lines 293 and BHK21. Treatment with dibutyryl cyclic adenosine- or guanosine monophosphate (cAMP, cGMP) did not alter the level of Cx45 phosphorylation, in either Cx45 transfectants or in 293 or BHK21 cells. The addition of the tumor-promoting agent phorbol 12-myristate 13-acetate (TPA) led to an increased 32P phosphate incorporation into the Cx45 protein in transfected cells. The Cx45 protein was found in homogenates of embryonic brain, kidney, and skin, as well as of adult lung. In kidney of four-day-old mice, Cx45 was detected in glomeruli and distal tubules, whereas connexin32 and -26 were coexpressed in proximal tubules. No connexin43 protein was detected in proximal tubules. No connexin43 protein was detected in renal tubules and glomeruli at this stage of development. Our results suggest that cells in proximal and distal tubules are interconnected by gap junction channels made of different connexin proteins. The Cx45 antibodies characterized in this paper should be useful for investigations of Cx45 in renal gap junctional communication.