The soluble guanylate cyclase stimulator riociguat and the soluble guanylate cyclase activator cinaciguat exert no direct effects on contractility and relaxation of cardiac myocytes from normal rats.

In cardiovascular diseases, reduced responsiveness of soluble guanylate cyclase (sGC) to nitric oxide (NO) upon long-term application has led to the development of NO-independent sGC stimulators (heme-dependent) and sGC activators (heme-independent). Any direct inotropic or lusitropic effects of these compounds on isolated cardiac myocytes, however, remain to be elucidated. Here, we analyzed the dose-dependent effects of clinical relevant concentrations (10(-10)-10(-5) M) of the sGC activator cinaciguat and the sGC stimulator riociguat on the contraction, relaxation, and calcium transients of isolated field-stimulated cardiac myocytes from healthy rats. For comparison, we used isoproterenol, which induced a dose-dependent significant increase in cell contractility, relaxation, and calcium transients, verapamil that significantly decreased these parameters (both at 10(-9)-10(-5) M) and 8-(4-Chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP) that induced a negative inotropic effect at 10(-5) M accompanied by a slight increase in relaxation. In contrast, neither cinaciguat nor riociguat significantly influenced any measured parameters. Furthermore, isoproterenol significantly increased intracellular cAMP levels that were not influenced by cinaciguat or riociguat (all at 10(-6) M). Otherwise, riociguat and cinaciguat (both at 10(-6) M) significantly enhanced intracellular cGMP generation. This accumulation was significantly augmented by cinaciguat in the presence of the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 25 µM), whereas ODQ blocked cGMP generation by riociguat. However, blocking of sGC did not influence cell contractility. Our results demonstrate that, in isolated cardiac myocytes from healthy rats, the increase in cGMP levels induced by cinaciguat and riociguat at clinical relevant concentrations is not associated with acute direct effects on cell contraction and relaxation.

High-density lipoproteins reduce palmitate-induced cardiomyocyte apoptosis in an AMPK-dependent manner.

Palmitate has been implicated in the induction of cardiomyocyte apoptosis via reducing the activity of 5' AMP-activated protein kinase (AMPK). We sought to evaluate whether high-density lipoproteins (HDLs), known for their cardioprotective features and their potential to increase AMPK activity, can reduce palmitate-induced cardiomyocyte apoptosis and whether this effect is AMPK-dependent. Therefore, cardiomyocytes were isolated from adult Wistar rat hearts via perfusion on a Langendorff-apparatus and cultured in free fatty acid-free BSA control medium or 0.5 mM palmitate medium in the presence or absence of HDL (5 µg protein/ml) with or without 0.1 µM of the AMPK-inhibitor compound S for the analysis of Annexin V/propidium, genes involved in apoptosis and fatty acid oxidation, and cardiomyocyte contractility. We found that HDLs decreased palmitate-induced cardiomyocyte apoptosis as indicated by a reduction in Annexin V-positive cardiomyocytes and an increase in Bcl-2 versus Bax ratio. Concomitantly, HDLs increased the palmitate-impaired expression of genes involved in fatty acid oxidation. Furthermore, HDLs improved the palmitate-impaired cardiomyocyte contractility. All effects were mediated in an AMPK-dependent manner, concluding that HDLs reduce palmitate-induced cardiomyocyte apoptosis, resulting in improved cardiomyocyte contractility through a mechanism involving AMPK.

Measurement of antibody effects on cellular function of isolated cardiomyocytes.

Dilated cardiomyopathy (DCM) is one of the main causes for heart failure in younger adults. Although genetic disposition and exposition to toxic substances are known causes for this disease in about one third of the patients, the origin of DCM remains largely unclear. In a substantial number of these patients, autoantibodies against cardiac epitopes have been detected and are suspected to play a pivotal role in the onset and progression of the disease. The importance of cardiac autoantibodies is underlined by a hemodynamic improvement observed in DCM patients after elimination of autoantibodies by immunoadsorption. A variety of specific antigens have already been identified and antibodies against these targets may be detected by immunoassays. However, these assays cannot discriminate between stimulating (and therefore functionally effective) and blocking autoantibodies. There is increasing evidence that this distinction is crucial. It can also be assumed that the targets for a number of cardiotropic antibodies are still unidentified and therefore cannot be detected by immunoassays. Therefore, we established a method for the detection of functionally active cardiotropic antibodies, independent of their respective antigen. The background for the method is the high homology usually observed for functional regions of cardiac proteins in between mammals. This suggests that cardiac antibodies directed against human antigens will cross-react with non-human target cells, which allows testing of IgG from DCM patients on adult rat cardiomyocytes. Our method consists of 3 steps: first, IgG is isolated from patient plasma using sepharose coupled anti-IgG antibodies obtained from immunoadsorption columns (PlasmaSelect, Teterow, Germany). Second, adult cardiomyocytes are isolated by collagenase perfusion in a Langendorff perfusion apparatus using a protocol modified from previous works. The obtained cardiomyocytes are attached to laminin-coated chambered coverglasses and stained with Fura-2, a calcium-selective fluorescent dye which can be easily brought into the cell to observe intracellular calcium (Ca(2+)) contents. In the last step, the effect of patient IgG on the cell shortening and Ca(2+) transients of field stimulated cardiomyocytes is monitored online using a commercial myocyte calcium and contractility monitoring system (IonOptix, Milton, MA, USA) connected to a standard inverse fluorescent microscope.

Immunoadsorption in dilated cardiomyopathy: long-term reduction of cardiodepressant antibodies.

BACKGROUND: Disturbances of humoral immunity have been described in dilated cardiomyopathy (DCM), and a number of antibodies against cardiac cell proteins have been identified. Previous studies showed that immunoadsorption therapy with subsequent IgG substitution (IA/IgG) enhances cardiac function, and that removal of cardiodepressant antibodies may represent one essential mechanism of this therapy. The long-term effect of IA/IgG on the level of cardiodepressant antibodies remains to be elucidated. METHODS: A total of 17 patients with DCM were observed up to 12 months after IA/IgG. Echocardiographic measurements were performed at baseline, 3, 6 and 12 months after therapy. Cardiodepressant antibodies were detected by incubation of rat cardiomyocytes with purified patients' IgG and recording of contractility and Ca(2+) ratio. RESULTS: In contrast to patients without cardiodepressant antibodies before IA/IgG, patients with negative inotropic antibodies showed an improvement of left ventricular ejection fraction (LVEF) from 33.8 +/- 1.7% to 44.7 +/- 2.7%; 44.5 +/- 2.3% and 51.8 +/- 1.7% after 3, 6 and 12 months (P < 0.001 vs. baseline, P < 0.05 vs. LVEF of non-cardiodepressant group). Immediately after IA/IgG therapy, no cardiodepressant effects of patients' IgG on isolated cardiomyocytes were detectable, and this effect remained diminished until 6 months after IA/IgG (P < 0.001 for contractility and Ca(2+) ratio). Compared with the levels after 3 and 6 months, cardiodepressant antibodies reoccured after 12 months (P = 0.067 for contractility, P < 0.05 for Ca(2+) ratio vs. 6 months after IA/IgG). However, the negative inotropic reaction is still diminished compared with the reaction before IA/IgG. CONCLUSION: IA/IgG therapy induces long-term reduction of negative inotropic antibodies. After 12 months, however, re-increase of negative inotropic antibodies cannot be excluded.