Klaus-Dieter Schlüter

Cardiovascular actions of parathyroid hormone and parathyroid hormone-related peptide

Cardiovascular cells (cardiomyocytes and smooth muscle cells) are target cells for parathyroid hormone (PTH) and the structurally related peptide parathyroid hormone-related peptide (PTH-rP). PTH activates protein kinase C (PKC) of cardiomyocytes via a PKC activating domain previously identified on chondrocytes. Activation of PKC leads to hypertrophic growth and re-expression of fetal type proteins in cardiomyocytes. This hypertrophic effect of PTH might contribute to left ventricular hypertrophy in hemodialysis patients with secondary hyperparathyroidism. PTH-rP is expressed in cardiovascular cells (endothelial cells and smooth muscle cells). It does not mimic the above described actions of PTH but exerts effects of its own on cardiomyocytes. These effects involve activation of protein kinase A, via a N-terminal domain distinct from that identified on PTH, and activation of PKC, via a C-terminally located domain distinct from that found on PTH. On smooth muscle cells PTH and PTH-rP reduce the influence of extracellular calcium, through cAMP-dependent mechanisms. These inhibitory effects on voltage-dependent L-type calcium channels of smooth muscle cells cause vasorelaxation. Present studies concerning cardiovascular actions of either PTH and PTH-rP suggest that increased plasma levels of PTH and PTH-rP influence cardiomyocyte and smooth muscle cell physiology. It can be assumed that PTH-rP acts as a paracrine or autocrine modulator in heart and vessels.

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.

Role of phosphatidylinositol 3-kinase activation in the hypertrophic growth of adult ventricular cardiomyocytes

OBJECTIVE The present study investigated whether activation of phosphatidylinositol 3-kinase (PI3-kinase) is involved in the stimulation of hypertrophic growth of adult ventricular cardiomyocytes under alpha- or beta-adrenoceptor stimulation. METHODS Adult ventricular rat cardiomyocytes were used either directly after isolation (day 1 culture) or after cultivation for 6 days in presence of 20% fetal calf serum (day 7 culture). PI3-kinase activity was determined in extracts of cardiomyocytes after immunoprecipitation with an antibody against the p85 subunit of PI3-kinase. The influence of PI3-kinase inhibition on myocardial growth was determined using the specific PI3-kinase inhibitors wortmannin and LY294002. RESULTS In day 1 cultures alpha-adrenoceptor stimulation, but not beta-adrenoceptor stimulation caused activation of PI3-kinase. In response to alpha-adrenoceptor stimulation but not beta-adrenoceptor stimulation an acceleration of protein synthesis (incorporation of 14C-phenylalanine) and an increase in the total masses of cellular protein and RNA was observed. In these cultures inhibition of PI3-kinase attenuated the acceleration of protein synthesis and the increase in cellular masses of protein or RNA in response to alpha-adrenoceptor stimulation. In day 7 cultures alpha- and beta-adrenoceptor stimulation caused activation of PI3-kinase and increased protein synthesis. In these cultures inhibition of PI3-kinase attenuated the growth response to alpha- and beta-adrenoceptor stimulation. CONCLUSIONS PI3-kinase activation via protein kinase C-dependent or cAMP-dependent pathways is required for hypertrophic growth of adult cardiomyocytes.

Early response kinase and PI 3-kinase activation in adult cardiomyocytes and their role in hypertrophy

The present study investigated the role of early response kinase (ERK) and phosphatidylinositol 3 (PI 3)-kinase in ventricular cardiomyocytes from adult rat for the hypertrophic response to α-adrenoceptor stimulation. Parameters of the hypertrophic response were stimulation of protein synthesis and induction of creatine kinase BB. The α-adrenoceptor agonist phenylephrine (10 μmol/l) activated ERK2 and PI 3-kinase. The protein kinase C inhibitor bisindolylmaleimide (5 μmol/l) and the mitogen-activated protein kinase kinase inhibitor PD-98059 (10 μmol/l) but not the tyrosine kinase inhibitor genistein (100 μmol/l) blocked ERK2 activation. Inhibition of ERK2 activation abolished induction of creatine kinase BB by phenylephrine but not the increase in protein synthesis. The PI 3-kinase inhibitor wortmannin (100 nmol/l) blocked protein synthesis under α-adrenoceptor stimulation but did not interfere with ERK2 activation. Inhibition of the ERK2 pathway with PD-98059 did not affect PI 3-kinase activation. We conclude that ERK2- and PI 3-kinase-dependent pathways represent two mutually exclusive ways of signaling that lead to different aspects of the hypertrophic response to α-adrenoceptor stimulation.The present study investigated the role of early response kinase (ERK) and phosphatidylinositol 3 (PI 3)-kinase in ventricular cardiomyocytes from adult rat for the hypertrophic response to alpha-adrenoceptor stimulation. Parameters of the hypertrophic response were stimulation of protein synthesis and induction of creatine kinase BB. The alpha-adrenoceptor agonist phenylephrine (10 micromol/l) activated ERK2 and PI 3-kinase. The protein kinase C inhibitor bisindolylmaleimide (5 micromol/l) and the mitogen-activated protein kinase kinase inhibitor PD-98059 (10 micromol/l) but not the tyrosine kinase inhibitor genistein (100 micromol/l) blocked ERK2 activation. Inhibition of ERK2 activation abolished induction of creatine kinase BB by phenylephrine but not the increase in protein synthesis. The PI 3-kinase inhibitor wortmannin (100 nmol/l) blocked protein synthesis under alpha-adrenoceptor stimulation but did not interfere with ERK2 activation. Inhibition of the ERK2 pathway with PD-98059 did not affect PI 3-kinase activation. We conclude that ERK2- and PI 3-kinase-dependent pathways represent two mutually exclusive ways of signaling that lead to different aspects of the hypertrophic response to alpha-adrenoceptor stimulation.

Regulation of growth in the adult cardiomyocytes

Cardiomyocytes of adult myocardium increase their cellular mass in response to growth stimuli. They undergo hypertrophic growth but they do not proliferate in contrast to immature cardiomy‐ocytes. Growth stimuli of the adult cardiomyocytes include classical growth hormones, various neuroendocrine factors, and the increase in mechanical load. The signal transduction of α1‐adrenoceptor stimulation has been investigated in greatest detail and may therefore be taken as a reference for other humoral stimuli. It involves the activation of protein kinase C (PKC) and, downstream of PKC activation, of two separate signaling pathways, one including the mitogen‐activated protein kinase and another including PI3‐kinase and p70s6k as key steps. Activation of the first pathway leads to re‐expression of fetal genes, activation of the second pathway to a general activation of protein synthesis, and cellular growth. In neonatal cardiomyocytes, mechanical stretch causes growth by an activation of an autocrine mechanism including angiotensin II and endothelin. This mechanism does not operate, however, in adult cardiomyocytes. A mechanism of mechanotransduction has not yet been identified on adult cardiomyocytes but integrins may play a part. In microgravity, the scenario of myocardial growth stimulation is altered. On the systemic level, there are changes in hemodynamic and neuroendocrine regulation that exert indirect effects on the myocardium. Microgravity may also exert a direct cellular effect by the absence of a constant gravitational load component.—Schlüter, K.‐D., Piper, H. M. Regulation of growth in the adult cardiomyocytes. FASEB J. 13 (Suppl.), S17–S22 (1999)

Induction of necrosis but not apoptosis after anoxia and reoxygenation in isolated adult cardiomyocytes of rat

OBJECTIVES Apoptosis is one feature of myocardial damage after ischemia-reperfusion, but the causes for its induction are unclear. The present study was undertaken to investigate whether apoptosis in cardiomyocytes is directly initiated by their sub-lethal injury that results from ischemia-reperfusion. METHODS Ischemia was simulated on isolated ventricular cardiomyocytes of adult rats by anoxia in a glucose free medium, pH 6.4. Induction of apoptosis was detected by (1) DNA laddering of genomic DNA, (2) TUNEL positive cells (terminal deoxynucleotidyl transferase-mediated-UTP nick end labelling) and (3) annexinV-fluorescein isothiocyanate (annexinV-FITC) binding to cells under exclusion of propidium iodide. Necrotic cells were identified by (1) staining with both annexinV-FITC and propidium iodide, (2) unspecific DNA degradation and (3) enzyme release. RESULTS Simulated ischemia caused a > 75% loss of high-energy phosphates within 2 h, which was reversible upon reoxygenation at pH 7.4. Even after 18 h of simulated ischemia, creatine phosphate contents recovered to 55.2 +/- 7.3% of control within 1 h. Apoptosis could be induced by UV irradiation (80 J/m2), H2O2 and the NO-donor N2-acetyl-S-nitroso-D,L-penicillinaminamide. In contrast to this, simulated ischemia and reoxygenation could not induce apoptosis in the cells, but with prolonged ischemia more cells became necrotic. After 18 hours of simulated ischemia and 4 h of reoxygenation 41.2 +/- 10.2% myocytes were necrotic (vs. 6.3 +/- 4.4% of control) and only 1.7 +/- 0.5% (vs. 8.7 +/- 4.6% of control) were apoptotic. The percentage of necrotic cells correlated with an increase in lactate dehydrogenase release from 9.9 +/- 0.6% (of total activity) of normoxic controls to 37.9 +/- 5.1% after 18 h of simulated ischemia and 12 h of reoxygenation. CONCLUSIONS Simulated ischemia-reoxygenation causes necrosis of isolated cardiomyocytes but is not sufficient for induction of apoptosis.

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.

Interplay between Ca2+ cycling and mitochondrial permeability transition pores promotes reperfusion-induced injury of cardiac myocytes

Uncontrolled release of Ca2+ from the sarcoplasmic reticulum (SR) contributes to the reperfusion‐induced cardiomyocyte injury, e.g. hypercontracture and necrosis. To find out the underlying cellular mechanisms of this phenomenon, we investigated whether the opening of mitochondrial permeability transition pores (MPTP), resulting in ATP depletion and reactive oxygen species (ROS) formation, may be involved. For this purpose, isolated cardiac myocytes from adult rats were subjected to simulated ischemia and reperfusion. MPTP opening was detected by calcein release and by monitoring the ΔΨm. Fura‐2 was used to monitor cytosolic [Ca2+]i or mitochondrial calcium [Ca2+]m, after quenching the cytosolic compartment with MnCl2. Mitochondrial ROS [ROS]m production was detected with MitoSOX Red and mag‐fura‐2 was used to monitor Mg2+ concentration, which reflects changes in cellular ATP. Necrosis was determined by propidium iodide staining. Reperfusion led to a calcein release from mitochondria, ΔΨm collapse and disturbance of ATP recovery. Simultaneously, Ca2+ oscillations occurred, [Ca2+]m and [ROS]m increased, cells developed hypercontracture and underwent necrosis. Inhibition of the SR‐driven Ca2+ cycling with thapsigargine or ryanodine prevented mitochondrial dysfunction, ROS formation and MPTP opening. Suppression of the mitochondrial Ca2+ uptake (Ru360) or MPTP (cyclosporine A) significantly attenuated Ca2+ cycling, hypercontracture and necrosis. ROS scavengers (2‐mercaptopropionyl glycine or N‐acetylcysteine) had no effect on these parameters, but reduced [ROS]m. In conclusion, MPTP opening occurs early during reperfusion and is due to the Ca2+ oscillations originating primarily from the SR and supported by MPTP. The interplay between Ca2+ cycling and MPTP promotes the reperfusion‐induced cardiomyocyte hypercontracture and necrosis. Mitochondrial ROS formation is a result rather than a cause of MPTP opening.

Adult Ventricular Cardiomyocytes

Isolated cardiomyocytes are a prerequisite to study the biology of cardiomyocytes. Efficient isolation is difficult, as these cells adhere firmly together in the heart and do not divide. Therefore, any experiment is restricted to the amount of calcium-tolerant, rod-shaped cardiomyocytes that can be initially isolated from the heart. This chapter gives detailed instructions on how ventricular cardiomyocytes can be isolated from an intact adult heart. The method is based on the principle of calcium-free perfusion with collagenase supplementation to disrupt cell-cell contacts in the heart, isolation and purification of cardiomyocytes from other cell types, and, finally, re-establishing a physiological cellular calcium concentration. The chapter also summarizes some commonly used adaptations to isolate cardiomyocytes from species different from rat.

Central role for ornithine decarboxylase in β-adrenoceptor mediated hypertrophy

Objective: TGF-β stimulation of cardiac myocytes induces a hypertrophic responsiveness to β-adrenoceptor stimulation. This study investigates whether this β-adrenoceptor mediated effect depends on induction of ornithine decarboxylase (ODC). Methods: Isolated adult ventricular cardiomyocytes from rats were used as an experimental model. Cells were either cultured in 20% (v/v) FCS to activate autocrine released TGF-β or used without pre-treatment. The hypertrophic response was characterized by an increased 14C-phenylalanine incorporation, RNA and protein mass or by an increased expression of atrionatriurectic factor and ODC. The results on cell cultures were compared to those achieved by isoprenaline perfused mice hearts from transgenic mice overexpressing TGF-β1. Results: ODC activity and expression increased within 2 h in TGF-β1 pre-treated cells under isoprenaline. In the presence of ODC inhibitors (α-methylornithine or difluoromethylornithine) this increase remained absent and the increases in 14C-phenylalanine incorporation, protein and RNA mass under isoprenaline were abolished. In cells not exposed to TGF-β no induction of ODC was observed. Isoprenaline also induced ODC in isolated perfused ventricles from transgenic mice overexpressing TGF-β1, but not in ventricles from their nontransgenic counterparts. Conclusions: This study shows first, a pivotal role for ODC induction in the hypertrophic response of cardiomyocytes to β-adrenoceptor stimulation and second, that ODC induction in vivo and in vitro requires pre-treatment of cardiomyocytes with TGF-β. It is concluded that TGF-β induces a hypertrophic responsiveness to β-adrenoceptor stimulation that is characterized by ODC induction.