Sibylle Wenzel

Redox-sensitive intermediates mediate angiotensin II-induced p38 MAP kinase activation, AP-1 binding activity, and TGF-β expression in adult ventricular cardiomyocytes

Cardiac hypertrophy as an adaptation to increased blood pressure leads to an increase in ventricular expression of transforming growth factor β (TGF‐β), probably via the renin‐angiotensin system. We studied in vivo to determine whether angiotensin II affects TGF‐β expression independent from mechanical effects caused by the concomitant increase in blood pressure and in vitro intracellular signaling involved in angiotensin II‐dependent TGF‐β1 induction. In vivo, the AT1 receptor antagonist losartan, but not reduction of blood pressure by hydralazine, inhibited the increase in TGF‐β1 expression caused by angiotensin II. In vitro, angiotensin II caused an induction of TGF‐β1 expression in adult ventricular cardiomyocytes and induced AP‐1 binding activity. Transfection with “decoys” directed against the binding site of AP‐1 binding proteins inhibited the angiotensin II‐dependent TGF‐β induction. Angiotensin II induced TGF‐β expression in a p38‐MAP kinase‐dependent way. p38‐MAP kinase activation was diminished in presence of the antioxidants or diphenyleneiodium chloride, or by pretreatment with antisense nucleotides directed against phox22 and nox, components of smooth muscle type NAD(P)H oxidase. Thus, our study identifies a previously unrecognized coupling of cardiac AT receptors to a NAD(P)H oxidase complex similar to that expressed in smooth muscle cells and identifies p38‐MAP kinase activation as an important downstream target.

Expression, Release, and Biological Activity of Parathyroid Hormone–Related Peptide From Coronary Endothelial Cells

Ventricular cardiomyocytes have previously been identified as potential target cells for parathyroid hormone-related peptide (PTHrP). Synthetic PTHrP peptides exert a positive contractile effect. Because systemic PTHrP levels are normally negligible, this suggests that PTHrP is expressed in the ventricle and acts as a paracrine mediator. We investigated the ventricular expression of PTHrP and its expression in cultured cells isolated from the ventricle, studied the release of PTHrP from hearts and cultures, and investigated whether this authentic PTHrP mimics the biological effects previously described for synthetic PTHrP on ventricular cardiomyocytes. We found PTHrP expressed in ventricles of neonatal and adult rat hearts. In cells isolated from adult hearts, we found PTHrP expression exclusively in coronary endothelial cells but not in cardiomyocytes. The latter, however, are target cells for PTHrP. PTHrP was released from isolated perfused hearts during hypoxic perfusion and from cultured coronary endothelial cells under energy-depleting conditions. This PTHrP was biologically active; ie, it exerted a positive contractile and lusitropic effect on cardiomyocytes. Authentic PTHrP was glycosylated and showed a slightly higher potency than synthetic PTHrP. These results suggest that PTHrP is an endothelium-derived modulator of ventricular function.

Lack of Endothelial Nitric Oxide Synthase–Derived Nitric Oxide Formation Favors Hypertrophy in Adult Ventricular Cardiomyocytes

Reduced activity and expression of endothelial NO synthase (eNOS) is observed in cardiomyocytes from pressure-overloaded hearts with heart failure. The present study was aimed to investigate whether reduced eNOS-derived NO production contributes to the hypertrophic growth and phenotype of these cardiomyocytes. Cultured ventricular cardiomyocytes from adult rats were exposed to N&ohgr;-nitro-l-arginine (l-NNA) to inhibit global NO formation, and cultured cardiomyocytes derived from eNOS-deficient mice were used as a model of genetic knockout of eNOS. Cell growth, formation of oxygen-derived radicals (reactive oxygen species [ROS]), activation of p38 mitogen-activated protein (MAP) kinase phosphorylation, and cytokine expression in cardiomyocytes were investigated. l-NNA caused a concentration-dependent acceleration of the rate of protein synthesis and an increase in cell size. This effect was sensitive to p38 MAP kinase inhibition or antioxidants. l-NNA induced a rapid increase in ROS formation, subsequent activation of p38 MAP kinase, and p38 MAP kinase–dependent increases in the expression of transforming growth factor-&bgr; and tumor necrosis factor-&agr;. Similar changes (increased ROS formation, p38 MAP kinase phosphorylation, and cytokine induction) were also observed in cardiomyocytes derived from eNOS+/+ mice when exposed to l-NNA. Cardiomyocytes from eNOS−/− mice displayed higher p38 MAP kinase phosphorylation and cytokine expression under basal conditions, but neither these 2 parameters nor ROS formation were increased in the presence of l-NNA. In conclusion, our data support the hypothesis that reduced eNOS activity in cardiomyocytes contributes to the onset of myocardial hypertrophy and increased cytokine expression, which are involved in the transition to heart failure.

New insights into paracrine mechanisms of human cardiac progenitor cells.

Cardiac progenitor cells (CPCs) have been shown to promote cardiac regeneration in vivo. Understanding the function of CPCs is essential for further implementation of these cells in the treatment of cardiac diseases. The present study tested the hypothesis that adult CPC exert paracrine effects that lead to an improvement in the functional characteristics of cardiomyocytes. This study also investigated whether aging (we included patients aged between 4 months and 81 years) has any effect on the paracrine mechanisms of CPC.

Controlling cardiomyocyte length: the role of renin and PPAR-γ

AIMS Renin and peroxisome proliferator-activated receptor (PPAR-γ) interact directly with cardiomyocytes and influence protein synthesis. We investigated their effects and interaction on the size of cardiomyocytes. METHODS AND RESULTS Effects of renin and PPAR-γ activation were studied in cultured adult rat ventricular cardiomyocytes, transgenic mice with a cardiomyocyte-restricted knockout of PPAR-γ, and transgenic rats overexpressing renin, TGR(mRen2)27. The length and width of cardiomyocytes were analysed 24 h after administration of factors. Renin caused an unexpected effect on the length of cardiomyocytes that was inhibited by mannose-6-phosphate and monensin, but not by administration of glucose-6-phosphate. Endothelin-1 used as a classical pro-hypertrophic agonist increased cell width but not cell length. Renin caused an activation of p38 and p42/44 mitogen-activated protein (MAP) kinases. The latter activation was impaired by mannose-6-phosphate. Inhibition of p42/44 but not of p38 MAP kinase activation attenuated the effect of renin on cell length. In contrast, activation of PPAR-γ reduced cell length. Feeding wild-type mice with pioglitazone, a PPAR-γ agonist, reduced cell length. Cardiomyocytes isolated from PPAR-γ knockout mice were longer, and their length was not affected by pioglitazone. Cardiomyocytes isolated from TGR(mRen2)27 rats were longer than those of non-transgenic littermates. Cell length was reduced by feeding these mice with pioglitazone. Pioglitazone affected cell length independent of blood pressure. CONCLUSION The length of cardiomyocytes is controlled by the activation of cardiac-specific mannose-6-phosphate/insulin-like growth factor II receptors and activation of PPAR-γ. This type of cell size modification differs from that of any other known pro-hypertrophic agonists.

Interleukin-6 Contributes to the Paracrine Effects of Cardiospheres Cultured from Human, Murine and Rat Hearts

Cardiosphere‐derived cells (CDCs) were cultured from human, murine, and rat hearts. Diluted supernatant (conditioned‐medium) of the cultures improved the contractile behavior of isolated rat cardiomyocytes (CMCs). This effect is mediated by the paracrine release of cytokines. The present study tested the hypothesis, that the cardiovascular state of the donor’s heart influences this effect on CMCs and tries to identify the responsible factors. CDCs were cultured from human tissue samples of cardiac surgery and from murine and rat hearts. The supernatants of cultured CDCs from hypertensive humans and rats showed a higher improvement of the contractile behavior of CMCs compared to CDCs of normotensive origin. Subsequently, the cytokine profile of the supernatants was analyzed. Among the cytokines elevated in supernatants originating from hypertensive humans or rats was Interleukin‑6. CDCs were also generated from Interleukin‑6−/−‐mice and their wildtype littermates. The supernatant of the cultured Interleukin‑6−/−‐CDCs had no effect on the contractile behavior, whereas the supernatant of the Interleukin‑6+/+‐CDCs showed a positive effect. To confirm the hypothesis that Interleukin‑6 contributes to the paracrine effects, CMCs were incubated with Interleukin‑6. It improved the contractile function in a concentration dependent way. Finally, the effect of the supernatant of cultured CDCs derived from a hypertensive human sample could be abolished by simultaneous incubation with a specific Interleukin‑6 antibody. CDCs release cytokines that improve the contractile behavior of CMCs. This effect is more intense in CDCs from hypertensive donors. Interleukin‑6 is involved in this phenomenon. J. Cell. Physiol. 229: 1681–1689, 2014.

TGF-β1 improves cardiac performance via up-regulation of laminin receptor 37/67 in adult ventricular cardiomyocytes

TGF-β1 plays an important role in cardiac fibrosis, apoptosis, induction of hypertrophy and contractile dysfunction. This study investigates whether TGF-β1 plays a role in laminin receptor 37/67 (37/67 LR)-dependent regulation of cardiac performance. Therefore, isolated adult cardiomyocytes were stimulated with TGF-β1, the expression of the 37/67 LR was determined and cell shortening was investigated on cells attached to a non-specific, serum-based attachment substrate or to specific, laminin-coated dishes. The role of the MAP kinases in TGF-β1-dependent induction of the 37/67 LR was examined by addition of PD98059, SB202190 and SP600125. Finally, the expression of receptor mRNA was investigated in transgenic mice constitutively over-expressing TGF-β1 and the relationship to distress score and lung wet weight-to-body weight was analysed. TGF-β1 induced a significant increase of the 37/67 LR mRNA and protein expression. The cytokine induced p38 MAP kinase and JNK, but not ERK. Inhibition of either p38 MAP kinase or JNK attenuated the TGF-β1-dependent increase in 37/67 LR expression. TGF-β1 induced a loss of cell shortening in cells attached to a non-specific substrate, but not in cells on a pre-coated laminin matrix. Inhibition of JNK attenuated the protective effect of laminin receptor up-regulation on cardiac performance. Inhibition of p38 MAP kinase attenuated the depressive effect of TGF-β1 on basal cell shortening. In transgenic mice over-expressing TGF-β1 a strong induction of laminin receptor expression attenuated the severeness of the mice’ symptoms. This study shows a new and protective role of TGF-β1-dependent up-regulation of the 37/67 LR in cardiomyocytes in cardiac remodelling with increased laminin expression.

Angiotensin II-dependent loss of cardiac function: Mechanisms and pharmacological targets attenuating this effect

Pharmacological inhibition of components of the renin‐angiotensin‐system is one of the major therapeutically options to treat patients with heart failure. This study hypothesized that angiotensin II (Ang II) directly depresses contractile function (cell shortening) by activation of transforming growth factor‐β1 (TGF‐β1). Moreover, we hypothesized that an inhibition of glycogen synthase kinase 3‐βGSK will compensate for this depressive effect by increasing SERCA2 expression. Isolated adult ventricular rat cardiomyocytes were used and cultured in the presence of Ang II (100 nM) for 24 h. Cell shortening and contractile dynamics were recorded at 2 Hz. Immunoblot techniques and gel mobility shift assays were used to demonstrate NFAT activation caused by inhibition of GSK and to demonstrate increases in the expression of SERCA2. Ang‐II caused a nearly 20% decrease in cell shortening. This Ang II‐dependent effect was mimicked by TGF‐β1 (10 ng/ml), attenuated by addition of aprotinin, that was used to block the proteolytic activation of TGF‐β1, or by application of a neutralizing antibody directed against TGF‐β1. Inhibition of GSK activated NFAT, increased SERCA2 expression and improved cell function. In conclusion, the study identified a paracrine mechanism for the Ang II‐dependent loss of cardiac function that occurs independently of hemodynamic changes. Furthermore, it characterized the differences between Ang II and α‐adrenoceptor stimulation with respect to the maintenance of cellular function explaining cellular events contributing to the difference between adaptive (physiological) and mal‐adaptive (patho‐physiological) hypertrophy. J. Cell. Physiol. 217: 242–249, 2008.

p38 MAP‐kinase in cultured adult rat ventricular cardiomyocytes: expression and involvement in hypertrophic signalling

Both α‐adrenoceptor‐ and β‐adrenoceptor‐stimulation lead to hypertrophic growth of the myocardium. But only β‐adrenoceptor‐stimulation requires the pre‐cultivation of cells with active TGF‐α. In order to define signalling molecules that are specifically involved in α‐adrenoceptor‐dependent hypertrophy, changes in expression and hypertrophic responsiveness during pre‐cultivation with TGF‐α were investigated. Isolated adult ventricular cardiomyocytes from rats were either cultured in 20% (v/v) foetal calf serum (FCS) to activate autocrine released TGF‐β or used without pre‐treatment. Protein synthesis was analysed by 14C‐phenylalanine incorporation. Expression of signalling molecules was determined by immunoblotting. During cultivation of cardiomyocytes with active TGF‐β only the expression of p38 MAP‐kinase increased. Subsequent stimulation of β‐adrenoceptors induced protein synthesis in a p38 MAP‐kinase‐dependent way. However, stimulation of β‐adrenoceptors activated p38 MAP‐kinase irrespective of pre‐treatment with TGF‐β. In the absence of this cytokine, hyperosmolarity or reconstitution of mechanical activity increased protein synthesis via p38 MAP‐kinase activation in freshly isolated cells. In conclusion, activation of p38 MAP‐kinase is a newly identified necessary signalling step required for β‐adrenoceptor induced hypertrophic growth. Like activation of adenyl cyclase, activation of p38 MAP‐kinase is up‐stream of the TGF‐β‐induced coupling to the regulation of protein synthesis. Reconstitution of mechanical activity mimics the co‐activation required and induced by TGF‐β.

Cell-Specific Effects of Nitric Oxide Deficiency on Parathyroid Hormone-Related Peptide (PTHrP) Responsiveness and PTH1 Receptor Expression in Cardiovascular Cells

The missing influence of estrogen on endothelial nitric oxide (NO) synthase often forms the basis for a worsening of the cardiac risk profile for women in postmenopause. Various studies have shown that decreasing estrogen levels also directly effect the expression of PTHrP and TGFbeta1. PTHrP is involved in the endothelium-dependent regulation of coronary resistance and cardiac function. The current study investigates to what extent chronic NO deficit affects the cardiac effects of PTHrP. NO deficit was achieved in female adult rats by feeding them the NO synthase inhibitor N-omega-nitro-L-arginine methyl ester over a period of 4 wk. Isolated hearts of the conditioned animals were investigated in Langendorff technique and perfused for 3 min with 100 nM PTHrP. The contraction behavior of isolated cardiomyocytes was registered in a cell-edge detection system. Hearts from untreated animals displayed a significant drop in left ventricular developed pressure and a pronounced increase in heart rate in consequence of short term PTHrP stimulation. In hearts from NO-deficient rats PTHrP no longer affected the inotropy and chronotropy. The vasodilating effect of PTHrP on coronary vessels was, however, independent of the NO level. These changes were accompanied by a differing expression of the PTH1 receptor. TGFbeta1 was identified as an important mediator for the regulation of the PTH1 receptor in myocytic but not endothelial cells. These results indicate that chronic NO deficit down-regulates the PTH1 receptor in a TGFbeta1-dependent way. These findings are important with respect to the relatively new therapy of postmenopausal osteoporosis with PTHrP analogs.