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Aims Disturbance of mitochondrial function significantly contributes to the myocardial injury that occurs during reperfusion. Increasing evidence suggests a role of intra-mitochondrial cyclic AMP (cAMP) signaling in promoting respiration and ATP synthesis. Mitochondrial levels of cAMP are controlled by type 10 soluble adenylyl cyclase (sAC) and phosphodiesterase 2 (PDE2), however their role in the reperfusion-induced injury remains unknown. Here we aimed to examine whether sAC may support cardiomyocyte survival during reperfusion. Methods and results Adult rat cardiomyocytes or rat cardiac H9C2 cells were subjected to metabolic inhibition and recovery as a model of simulated ischemia and reperfusion. Cytosolic Ca2+, pH, mitochondrial cAMP (live-cell imaging), and cell viability were analyzed during a 15-min period of reperfusion. Suppression of sAC activity in cardiomyocytes and H9C2 cells, either by sAC knockdown, by pharmacological inhibition or by withdrawal of bicarbonate, a natural sAC activator, compromised cell viability and recovery of cytosolic Ca2+ homeostasis during reperfusion. Contrariwise, overexpression of mitochondria-targeted sAC in H9C2 cells suppressed reperfusion-induced cell death. Analyzing cAMP concentration in mitochondrial matrix we found that inhibition of PDE2, a predominant mitochondria-localized PDE isoform in mammals, during reperfusion significantly increased cAMP level in mitochondrial matrix, but not in cytosol. Accordingly, PDE2 inhibition attenuated reperfusion-induced cardiomyocyte death and improved recovery of the cytosolic Ca2+ homeostasis. Conclusion sAC plays an essential role in supporting cardiomyocytes viability during reperfusion. Elevation of mitochondrial cAMP pool either by sAC overexpression or by PDE2 inhibition beneficially affects cardiomyocyte survival during reperfusion.

Background Ischemic preconditioning ( IPC ) and ischemic postconditioning ( IP oC) are currently among the most efficient strategies protecting the heart against ischemia/reperfusion injury. However, the effect of IPC and IP oC on functional recovery following ischemia/reperfusion is less clear, particularly with regard to the specific receptor‐mediated signaling of the postischemic heart. The current article examines the effect of IPC or IP oC on the regulation and coupling of β‐adrenergic receptors and their effects on postischemic left ventricular function. Methods and Results The β‐adrenergic signal transduction was analyzed in 3‐month‐old Wistar rats for each of the intervention strategies (Sham, ischemia/reperfusion, IPC , IP oC) immediately and 7 days after myocardial infarction. Directly after the infarction a cardioprotective potential was demonstrated for both IPC and IP oC: the infarct size was reduced, apoptosis and production of reactive oxygen species were lowered, and the myocardial tissue was preserved. Seven days after myocardial ischemia, only IPC hearts showed significant functional improvement. Along with a deterioration in fractional shortening, IP oC hearts no longer responded adequately to β‐adrenergic stimulation. The stabilization of β‐adrenergic receptor kinase‐2 via increased phosphorylation of Mdm2 (an E3‐ubiquitin ligase) was responsible for desensitization of β‐adrenergic receptors and identified as a characteristic specific to IP oC hearts. Conclusions Immediately after myocardial infarction, rapid and transient activation of β‐adrenergic receptor kinase‐2 may be an appropriate means to protect the injured heart from excessive stress. In the long term, however, induction and stabilization of β‐adrenergic receptor kinase‐2, with the resultant loss of positive inotropic function, leads to the functional picture of heart failure.

Experimental and clinical studies demonstrated that postconditioning confers protection against myocardial ischemia/reperfusion injury. However the underlying cellular mechanisms responsible for the beneficial effect of postconditioning are still poorly understood. The aim of the present study was to examine the role of cytosolic and mitochondrial Ca(2+)-handling. For this purpose adult rat cardiomyocytes were subjected to simulated in vitro ischemia (glucose-free hypoxia at pH6.4) followed by simulated reperfusion with a normoxic buffer (pH7.4; 2.5 mmol/L glucose). Postconditioning, i.e., 2 repetitive cycles of normoxic (5s) and hypoxic (2.5 min) superfusion, was applied during the first 5 min of reoxygenation. Mitochondrial membrane potential (ΔΨm), cytosolic and mitochondrial Ca(2+) concentrations, cytosolic pH and necrosis were analysed applying JC-1, fura-2, fura-2/manganese, BCECF and propidium iodide, respectively. Mitochondrial permeability transition pore (MPTP) opening was detected by calcein release. Hypoxic treatment led to a reduction of ΔΨm, an increase in cytosolic and mitochondrial Ca(2+) concentration, and acidification of cardiomyocytes. During the first minutes of reoxygenation, ΔΨm transiently recovered, but irreversibly collapsed after 7 min of reoxygenation, which was accompanied by MPTP opening. Simultaneously, mitochondrial Ca(2+) increased during reperfusion and cardiomyocytes developed spontaneous cytosolic Ca(2+) oscillations and severe contracture followed by necrosis after 25 min of reoxygenation. In postconditioned cells, the collapse in ΔΨm as well as the leak of calcein, the increase in mitochondrial Ca(2+), cytosolic Ca(2+) oscillations, contracture and necrosis were significantly reduced. Furthermore postconditioning delayed cardiomyocyte pH recovery. Postconditioning by hypoxia/reoxygenation was as protective as treatment with cyclosporine A. Combining cyclosporine A and postconditioning had no additive effect. The data of the present study demonstrate that postconditioning by hypoxia/reoxygenation prevents reperfusion injury by limiting mitochondrial Ca(2+) load and thus opening of the MPTP in isolated cardiomyocytes. These effects seem to be supported by postconditioning-induced delay in pH recovery and suppression of Ca(2+) oscillations.

Since adenine nucleotide translocase 1 (ANT1) overexpression improved cardiac function in rats with activated renin-angiotensin system (RAS) and angiotensin II is known to enhance transforming growth factor β (TGFβ) signaling in cardiomyocytes, we assumed that ANT1 might modulate the classical TGFβ/SMAD pathway. We therefore investigated whether the cardioprotective effect of ANT1 overexpression suppresses TGFβ(1)-induced apoptosis, whether mitochondrial permeability transition pore (MPTP) regulation is involved, and SMAD signaling pathway is affected.Ventricular cardiomyocytes isolated from wild-type (WT) and ANT1 transgenic rats were treated with the apoptosis-inducing agent TGFβ(1) (1 ng/ml). TGFβ(1) treatment of WT cells enhanced the number of apoptotic cells by 31.8 ± 11.7% (p0.01 vs. WT) measured by chromatin condensation. Apoptosis was blocked by 1μM cyclosporine A and by ANT1 overexpression. The protecting effect of ANT1 overexpression on TGFβ(1)-induced apoptosis was verified by reduced caspase 3/7 activity and increased Bcl-2 expression. In addition, TGFβ(1) decreased mitochondrial membrane potential as measured by JC-1 staining by 18.0 ± 3.7% in WT cardiomyocytes, but only by 7.2 ± 2.8% (p0.05 vs. WT) in ANT1 cardiomyocytes. Cyclosporine A also attenuated the decline in mitochondrial membrane potential under TGFβ(1) in WT cardiomyocytes. Determination of MPTP opening by Calcein assay in isolated cardiomyocytes and calcium retention assay in isolated mitochondria revealed a reduced open probability of MPTP after ANT1 overexpression. In addition to the effects of ANT1 on MPTP opening we investigated if ANT1 may interfere with the classical TGFβ signaling pathway. Interestingly, ANT1-transgenic cardiomyocytes expressed less TGFβ receptor II than WT cells. However, SMAD2 phosphorylation was already enhanced without TGFβ(1) stimulation in these cells. Although no additional increase in SMAD2 phosphorylation was detectable after TGFβ(1) treatment, SMAD signaling was still responsive to TGFβ(1) indicated by an upregulation of SMAD7, a TGFβ(1) target protein.Heart-specific overexpression of ANT1 leads to a reduced apoptotic response to TGFβ(1) by preservation of the mitochondrial membrane potential, resistance to MPTP opening and altered TGFβ signaling.

Uncontrolled release of Ca(2+) 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 ΔΨ(m). Fura-2 was used to monitor cytosolic [Ca(2+)](i) or mitochondrial calcium [Ca(2+)](m), after quenching the cytosolic compartment with MnCl(2). Mitochondrial ROS [ROS](m) production was detected with MitoSOX Red and mag-fura-2 was used to monitor Mg(2+) concentration, which reflects changes in cellular ATP. Necrosis was determined by propidium iodide staining. Reperfusion led to a calcein release from mitochondria, ΔΨ(m) collapse and disturbance of ATP recovery. Simultaneously, Ca(2+) oscillations occurred, [Ca(2+)](m) and [ROS](m) increased, cells developed hypercontracture and underwent necrosis. Inhibition of the SR-driven Ca(2+) cycling with thapsigargine or ryanodine prevented mitochondrial dysfunction, ROS formation and MPTP opening. Suppression of the mitochondrial Ca(2+) uptake (Ru360) or MPTP (cyclosporine A) significantly attenuated Ca(2+) 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 Ca(2+) oscillations originating primarily from the SR and supported by MPTP. The interplay between Ca(2+) cycling and MPTP promotes the reperfusion-induced cardiomyocyte hypercontracture and necrosis. Mitochondrial ROS formation is a result rather than a cause of MPTP opening.

Intermedin, a novel member of the calcitonin/calcitonin gene-related peptide family identified from vertebrate genomes, may directly affect cardiac function but current studies revealed no clear picture. The aims of our study were to compare direct contractile effects of intermedin on cardiomyocytes to that on the whole organ and to investigate whether intermedin improves postischemic recovery independent of an effect on acute reperfusion injury. Isolated adult rat ventricular cardiomyocytes were electrically paced and cell shortening was monitored as a readout associated to cardiac performance. Calcium transients were analyzed by Fura-2AM loading of these cells. Isolated rat hearts were investigated by Langendorff perfusion under nonischemic conditions and after 45-min no-flow ischemia followed up by 30-min reperfusion prior to drug testing. Intermedin caused a positive contractile effect on cardiomyocytes that was mediated by protein kinase A activation and accompanied by improved calcium transients. In contrast, intermedin reduced left ventricular developed pressure in Langendorff-perfused rat hearts. This negative inotropic effect was attenuated by inhibition of nitric oxide synthesis. In postischemic hearts (impaired nitric oxide synthesis), the negative inotropic effect was attenuated but no positive inotropic effect occurred. However, intermedin caused robust vasodilation in nonischemic and postischemic hearts. Our findings suggest that the peptide binds preferentially to vascular cells in the intact organ. The loss of nitric oxide induction in postischemic hearts attenuates a negative inotropic effect of intermedin but does not improve cardiac performance independent of acute reperfusion injury.

Ischemia-induced apoptosis of endothelial cells may contribute to tissue injury, organ failure, and transplantation rejection. However, little is known about survival mechanisms capable to counteract endothelial apoptosis. This study investigated the potential role of an endogenous anti-apoptotic response elicited by transient hypoxia, capable to avert ongoing apoptosis in endothelial cells. Experiments were carried out in three different types of cultured endothelial cells (human umbilical vein, pig aorta, and from rat coronary microvasculature). As a pro-apoptotic challenge endothelial cells were cultured in serum-free medium and subjected to hypoxia for 2 h. We found that transient hypoxia reduced caspase 3 activation within 1 h of hypoxia. Accordingly, the number of apoptotic cells was reduced after 24 h of reoxygenation. This was true for all three cell types analyzed. Analysis of Akt and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathways revealed that hypoxia induced a transient activation of ERK 2 but not of Akt. ERK 2 phosphorylation preceded the phosphorylation of pro-apoptotic molecule Bad at Ser112, an inhibitory phosphorylation site specific for ERK. The protective effects of hypoxia regarding Bad phosphorylation, caspase 3 activation, and apoptosis were abolished by MEK 1/2 inhibitors, PD98059 or UO126, as well as by antisense oligonucleotides directed against ERK 1/2. Furthermore, inhibition of this pathway inhibited hypoxia-induced increase in mitochondrial membrane potential. The present study demonstrates that transient hypoxia induces a novel survival mechanism that protects endothelial cells against apoptosis. This endogenous process involves MEK/ERK-mediated inhibition of the pro-apoptotic molecule Bad and caspase 3.

Bicarbonate transport (BT) has been previously shown to participate in apoptosis induced by various stress factors. However, the precise role of BT in ischaemia-induced apoptosis is still unknown. To investigate this subject, rat coronary endothelial cells (EC) were exposed to simulated ischaemia (glucose free anoxia at Ph 6.4) for 2 hrs and cells undergoing apoptosis were visualized by nuclear staining or by determination of cas-pase- 3 activity. To inhibit BT, EC were either treated with the inhibitor of BT 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS, 300 μmol/l) or exposed to ischaemia in bicarbonate free, 4-(2-hydroxyethyl)-I-piperazi-neethanesulphonic acid (HEPES)-buffered medium. Simulated ischaemia in bicarbonate-buffered medium (Bic) increased caspase-3 activity and the number of apoptotic cell (23.7 + 1.4%versus 5.1 + 1.2% in control). Omission of bicarbonate during ischaemia further significantly increased caspase-3 activity and the number of apoptotic cells (36.7 1.7%). Similar proapoptotic effect was produced by DIDS treatment during ischaemia in Bic, whereas DIDS had no effect when applied in bicarbonate-free, HEPES-buffered medium (Hep). Inhibition of BT was without influence on cytosolic acidification during ischaemia and slightly reduced cytosolic Ca2+ accumulation. Initial characterization of the underlying mechanism leading to apoptosis induced by BT inhibition revealed activation of the mitochondrial pathway of apoptosis, i.e., increase of cytochrome C release, depolarization of mitochondria and translocation of Bax protein to mitochondria. In contrast, no activation of death receptor-dependent pathway (caspase-8 cleavage) and endoplasmic reticulum- dependent pathway (caspase-12 cleavage) was detected. In conclusion, BT plays an important role in ischaemia-induced apoptosis of coronary EC by suppression of mitochondria-dependent apoptotic pathway.

Parathyroid hormone-related peptide (PTHrP) was found to improve contractile function of stunned myocardium in pigs. The peptide is released from coronary endothelial cells during ischemia and significantly improves post-ischemic recovery. The present study was aimed to decide whether such an induction of contractile responsiveness of the heart requires co-activation of adjacent cells or is a genuine phenomenon of cardiomyocytes. A second aim of this study was to decide whether such an improvement is linked to depressed cell function in general or oxidative inhibition. Isolated adult ventricular cardiomyocytes from rats were constantly paced at 0.5 Hz for 10 min. Cells were exposed to a brief oxidative inhibition by addition of 0.5 mmol/l potassium cyanide (KCN) in the presence of glucose. Under these conditions, cells stopped beating after 280 s on average. 30 s before they stopped to beat, cells had already developed a reduction in cell shortening, maximal relaxation and contraction velocity. In the co-presence of PTHrP (1-34) (100 nmol/l) cells continued to beat regular and did not develop reduced cell shortening, irrespectively of oxidative inhibition. In a second attempt, cells were exposed to the NO donor SNAP (100 micromol/l) or 8-bromocGMP (1 mmol/l). As expected both agents reduced cell shortening significantly. This reduction in cell shortening was attenuated in co-presence of PTHrP, too. Finally, we investigated the effect of PTHrP on cell shortening at different extracellular concentrations of calcium. Although, PTHrP increased intracellular calcium at 2 and 5 mmol/l extracellular calcium, respectively, it improved cell shortening only at 5 mmol/l extracellular calcium. Thus, the beneficial effect of PTHrP on cell shortening was independent from intracellular calcium but dependent on the steepness of the calcium gradient between intra- and extracellular calcium. In conclusion, our study shows that PTHrP is able to improve cell shortening rapidly and directly irrespectively of the reason for the reduced cell function. Improved electromechanical coupling rather than intracellular calcium handling seems to be the most important mechanism.

Tuberoinfundibular peptide (TIP39) was initially identified as a neurotransmitter and agonist of the PTH2 receptor, which is expressed in the cardiovascular system. This study documents for the first time the cardiac expression and function of TIP39. Expression was analyzed via RT-PCR. Function was characterized on Langendorff-perfused rat hearts as left ventricular developed pressure (LVDP) and on isolated cells via a cell edge detection system. cGMP levels were detected with RIA. Tuberoinfundibular peptide (TIP39) mRNA was found to be constitutively expressed in coronary endothelium cells, isolated cardiomyocytes, ventricles, atria, and aorta. At first we investigated the vasodilatory properties of TIP39 (100 nM) in the presence of L-nitro-arginine (L-NA, 100 microM). Surprisingly, TIP39 had no vasodilatory effect but decreased LVDP by 35 +/- 7%. In the absence of L-NA, addition of TIP39 decreased LVDP by 8 +/- 2%. The PTH2 receptor antagonist Trp23-Tyr36-PTHrP (1-36, 100 nM) abolished this TIP39 effect in the presence of L-NA. The experiments with isolated cardiomyocytes provided similar results. TIP39 (10 nM) lowered the contraction amplitude by 6 +/- 3%. In the presence of L-NA (100 micromol/liter), TIP39 lowered the amplitude by 34 +/- 6%. cGMP concentration in cardiomyocytes was stimulated by TIP39 (10 nM) in the same range as by the nitric oxide (NO) donor SNAP (100 microM). In the presence of L-NA, this increase was abolished. These results suggest that an inhibition of endogenous NO production unmasks a profound negative inotropic effect of TIP39 that is mediated by an activation of the PTH2 receptor. The results obtained with isolated cardiomyocytes suggest that myocyte-derived NO, rather than vascular NO, is responsible for this effect. cGMP seems to be the downstream signal of produced NO.

The calcium-sensing receptor (CaR) is widely expressed throughout the entire cardiovascular system and is capable of activating signaling pathways in different cells. Alongside calcium, the CaR also responds to physiological polycations such as putrescine underlining a participation in physiological and pathophysiological processes. Here, we aimed to determine mechanisms as to how CaR activation affects the contractile responsiveness of ventricular cardiomyocytes under basal and stimulated conditions. For that purpose, cardiac myocytes from 3-month-old male Wistar rats were isolated, and the acute effects of an antagonist (NPS2390), agonists (putrescine and gadolinium), or of downregulation of the CaR by siRNA on cell shortening were recorded in a cell-edge-detection system. In addition, experiments were performed on muscle stripes and Langendorff preparations. Mechanistic insights were taken from calcium transients of beating fura-2 AM-loaded cardiomyocytes and western blots. Isolated ventricular cardiomyocytes constitutively express CaR. The expression in the atria is less pronounced. Acute inhibition of CaR reduced basal cell shortening of ventricular myocytes at nearly physiological levels of extracellular calcium. Inhibition of CaR strongly reduced contractility of ventricular muscle stripes but not of atria. Activation of CaR by putrescine and gadolinium influences the contractile responsiveness of isolated cardiomyocytes. Increased calcium mobilization from the sarcoplasmic reticulum via an IP3-dependent mechanism was responsible for amplified systolic calcium transients and a subsequent improvement in cell shortening. Alongside with these effects, activation of CaR increased relaxation velocity of the cells. In conclusion, ventricular CaR expression affects contractile parameters of ventricular heart muscle cells and modifies electromechanical coupling of cardiomyocytes.

Transforming growth factor β (TGFβ) expression is induced in the myocardium during transition from compensated hypertrophy to heart failure. In cardiomyocytes, stimulation with TGFβ results in restricted contractile function and enhanced apoptosis. Nitric oxide (NO) also induces apoptosis and influences cardiac function. Therefore, we wanted to know whether NO is causally involved in TGFβ-induced apoptosis. In isolated ventricular cardiomyocytes of adult rat incubation with TGFβ1 increased NO release which was inhibited by NOS inhibitor ETU but not with iNOS inhibitor (1400 W) or nNOS inhibitor (TFA). In addition, TGFβ-induced apoptosis was blocked with ETU and ODQ, but not with 1400 W or TFA. The consequent assumption that endothelial NOS is involved in TGFβ-induced NO formation and apoptosis was supported by increased phosphorylation of eNOS at serine 1177 and by the fact that TGFβ did not increase NO release in eNOS KO mice. Furthermore, TGFβ-induced apoptosis, NO formation, SMAD binding activity and SMAD2 phosphorylation were blocked by a TGFβ receptor antagonist, but only apoptosis and NO formation could be blocked with ETU. Expression of SMAD7 was increased after TGFβ stimulation and blocked with TGFβ receptor antagonist but not after blocking NO synthase with ETU. Conclusion: In cardiomyocytes TGFβ-induced apoptosis is mediated via TGFβ receptor activation that concomitantly activates SMAD transcription factors and the eNOS/NO/sGC pathway. Both of these pathways are needed for apoptosis induction by TGFβ. This reveals a new pathway of cardiac NO release and identifies NO as a possible contributor to heart failure progression mediated by TGFβ. J. Cell. Physiol. 226: 2683–2690, 2011. © 2010 Wiley-Liss, Inc.

ATP can differentially affect the micro- and macrovascular endothelial barrier. It has been shown that it can both increase and/or decrease macromolecule permeability of microvascular endothelial cells and microvessels, in vivo. We hypothesised that the barrier stabilising effect is mediated by ATP itself via P2 receptors, while barrier-disrupting effect is mediated by its metabolite adenosine via adenosine receptors. The effects of ATP, ADP, AMP and adenosine on barrier function were studied in cultured rat coronary microvascular endothelial monolayers (RCEC) in vitro, as well as in rat mesentery vessels, and in rat hearts in vivo. ATP and ADP showed a biphasic effect on permeability of RCEC monolayers with a reduction followed by a later increase in albumin permeability. The permeability decreasing effect of ATP was enhanced by ecto-nucleotidase inhibitor ARL67156 while permeability increasing effect was enhanced by apyrase, an extracellular ecto-nucleotidase. Moreover, the permeability increasing effect was abrogated by adenosine receptor antagonists, 8-phenyltheophylline (8-PT) and DMPX. Adenosine and adenosine receptor agonists 5'-(N-ethylcarboxamido)-adenosine (NECA), CGS21680, and R-PIA enhanced albumin permeability which was antagonised by 8-PT, A(1), and A(2) but not by A(3) receptor antagonists. Likewise, immunofluorescence microscopy of VE-cadherin and actin showed that NECA induces a disturbance of intercellular junctions. Pre-incubation of ATP antagonised the effects of NECA on permeability, actin cytoskeleton and intercellular junctions. Similar effects of the applied substances were observed in rat mesentery artery by determining the vascular leakage using intravital microscopy as well as in rat hearts by assessing myocardial water contents in vivo. In conclusion, the study demonstrates that in RCEC, ATP, ADP, and its metabolite adenosine play opposing roles on endothelial barrier function.

The effects of statin treatment in the setting of heart failure have already been shown. Nevertheless, there is little knowledge about its influence on adrenergic pathways in cardiomyocytes. Therefore, this study investigated the impact of cerivastatin on adrenoceptor-mediated signalling pathways in isolated adult ventricular cardiomyocytes. It focused on two endpoints: hypertrophic growth and TGFbeta expression. Cultured cardiomyocytes were used to study rac activation (analysed by its translocation into the membrane fraction), ROS formation (H 2 DCF fluorescence) and hypertrophic growth ( 14 C-phenylalanine incorporation). Alpha- and beta-adrenoceptor stimulation showed significant differences regarding rac activation, ROS formation, and p38 MAP kinase activation. Both alpha- and beta-adrenoceptor stimulation induced TGFbeta expression. Upon activation of alpha-adrenergic signalling – although ROS formation was not influenced by cerivastatin – TGFbeta expression decreased. Following beta stimulation, TGFbeta expression as well as rac and p38 MAP kinase activation were reduced after pre-treatment with cerivastatin. Statin treatment did not show any influence on hypertrophic growth. In summary, this study clearly demonstrates the ability of adrenoceptor stimulation to increase TGFbeta expression. One component of the beneficial effects of statin therapy on heart failure might therefore be due to a dominant reduction and inhibition of TGFbeta, which is involved in many pathophysiological processes in cardiomyocytes.

Aims 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. Methods and results The supernatant of CPC generated both from human and rat hearts—so called ‘conditioned cardiosphere medium’ improved the contractile behaviour of isolated adult cardiomyocytes in a concentration-dependent manner after incubation for 24 h and increased the SERCA/NCX ratio. The observed positive effects on contractile behaviour were independent of the CPC donors' age. Conditioned cardiosphere media also normalized angiotensin II-induced contractile dysfunction. Cytokines released by CPC into the media were detected by cytokine arrays. Conclusion The observed diversity of cytokines released by CPC needs to be further elucidated in detail. Nevertheless, CPC are a promising therapeutic approach in the field of cardiac disease. The methods described allow investigation of the underlying paracrine mechanisms in a standardized in vitro situation.

Heart failure is accompanied by electrolyte disturbance including reduced calcium and sodium in the extracellular milieu but increased calcium within cells, a phenomenon called "calcium paradox". Aldosteronism is considered as part of this disorder. Aldosterone antagonism is known to reduce cardiac mortality on top of standard therapies such as antagonism of the renin-angiotensin-system. However, the effect of aldosterone on cardiac function under basal conditions and conditions more closely related to those seen in heart failure remains elusive. In order to address this question the function of isolated cardiomyocytes was determined as unloaded cell shortening. Cardiomyocytes were isolated from adult rat hearts and cultured for 24 h in the presence of aldosterone. Thereafter, cell shortening was determined in cells that were electrically paced (0.5-2.0 Hz). The effect of aldosterone on cell shortening was investigated under basal and maximal inotropic stimulation, preincubation with angiotensin II and myocytes from spontaneously hypertensive rats. The composition of the culture medium was modified according to the extracellular milieu found in patients with end-stage heart failure. Aldosterone increased cell shortening in a frequency-dependent way under basal conditions and conditions of low calcium. It potentiated the effect of beta-adrenoceptor stimulation, increased the formation of oxygen radicals, and increased diastolic and systolic calcium. In conclusion, chronic exposure to aldosterone improves the function of cardiomyocytes under basal conditions and electrolyte disturbances that mimic the situation found in heart failure patients.

Ischemic preconditioning has a powerful protective potential against a reperfusion-induced injury of the post-ischemic myocardium. Cardiomyocyte hypercontracture, i.e. excessive cell shortening, is an essential mechanism of the reperfusion-induced injury. Rigor contracture, i.e. Ca(2+)-independent contracture, has been shown to be an import component of the reperfusion-induced hypercontracture. Since rigor contracture is dependent on the rapidity of the metabolic recovery during reoxygenation, we hypothesized that preconditioning of the cardiomyocyte mitochondria may improve mitochondrial function to restore the energy balance during the initial phase of reoxygenation and may thus prevent rigor contracture. For this purpose adult rat cardiomyocytes were exposed to anoxia with subsequent reoxygenation. For preconditioning, cells were pre-treated with the mitochondrial ATP-sensitive K(+) channel opener diazoxide. Pre-treatment with 100 micromol/l diazoxide significantly reduced the reoxygenation-induced hypercontracture of cardiomyocytes due to an attenuation of the Ca(2+)-independent rigor-type contracture, which was accompanied by an acceleration of the phosphocreatine resynthesis during the initial phase of reoxygenation. Treatment with the mitochondrial ATP-sensitive K(+) channel antagonist 5-hydroxydecanoate (500 micromol/l) during preconditioning phase abolished these protective effects. Similarly, partial suppression of the mitochondrial function with 100 micromol/l NaCN during the reoxygenation phase abolished the diazoxide effects. Finally, in isolated rat hearts, preconditioning with diazoxide prior to global ischemia significantly improved left ventricular function and attenuated hypercontracture during reperfusion. This effect could be abolished by the treatment with 100 micromol/l NaCN during reperfusion. Taken together, pharmacological preconditioning of cardiomyocytes with diazoxide protects against the reoxygenation-induced rigor hypercontracture due to an improvement of the energy recovery at the onset of reoxygenation.

Aims Ischaemia–reperfusion provokes barrier failure of the coronary microvasculature, impeding functional recovery of the heart during reperfusion. The aim of the present study was to investigate whether the stimulation of cGMP signalling by activation of soluble guanylyl cyclase (sGC) can reduce reperfusion-induced endothelial intercellular gap formation and to determine whether this is due to an influence on endothelial cytosolic Ca2+ homeostasis during reperfusion. Methods and results Experiments were performed with cultured coronary endothelial monolayers and isolated saline-perfused rat hearts. HMR1766 (1 µmol/L) or DEAnonoate (0.5 µmol/L) were used to activate sGC. After exposure to simulated ischaemic conditions, reperfusion of endothelial cells led to a pronounced increase in cytosolic calcium levels and intercellular gaps. Stimulation of cGMP signalling during reperfusion increased Ca2+ sequestration in the endoplasmic reticulum (ER) and attenuated the reperfusion-induced increase in cytosolic [Ca2+]. Phosphorylation of phospholamban was also increased, indicating a de-inhibition of the ER Ca2+ pump (SERCA). Reperfusion-induced intercellular gap formation was reduced. Reduction of myosin light chain phosphorylation indicated inactivation of the endothelial contractile machinery. Effects on cytsolic Ca2+ and gaps were abrogated by inhibition of cGMP-dependent protein kinase (PKG) with KT5823. In reperfused hearts, stimulation of cGMP signalling led to decreased oedema development. Conclusion sGC/PKG activation during reperfusion reduces reperfusion-induced endothelial intercellular gap formation by attenuation of cytosolic calcium overload and reduction of contractile activation in endothelial cells. This mechanism protects the heart against reperfusion-induced oedema.

Aims In patients with congestive heart failure, plasma parathyroid hormone (PTH) levels are positively associated with cardiac function. PTH, used to mobilize stem cells from the bone marrow after myocardial infarction, causes an increased left ventricular ejection fraction. The aim of this study was to investigate whether low but plasma-relevant concentrations of PTH directly influence the contractile properties of cardiomyocytes. Methods and results Isolated adult rat ventricular cardiomyocytes were exposed to PTH(1–34) or full-length PTH at picomolar concentrations for 24 h. Cell shortening was measured at 2 Hz as a cellular correlate of inotropic responsiveness. Intracellular calcium was measured in Fura-AM-loaded cells. PTH(1–3) (20–200 pM) and full-length PTH (200 pM) increased cell shortening within 24 h. PTH had no effect on cell size, but resting and peak systolic calcium concentrations were elevated. The beneficial effect of PTH was mediated via its cAMP/protein kinase A-activating domain and attenuated by addition of a protein kinase A inhibitor. In contrast, PTH peptides representing a protein kinase C-activating domain but not a cAMP/protein kinase A-activating domain or peptides that represent none of these domains had no effect on cell shortening. The effect of PTH on cell shortening was strong at low concentrations of extracellular calcium but declined at higher calcium concentrations. PTH downregulated the expression of the calcium sensing receptor, a receptor known to antagonize the action of PTH on calcium transport. Furthermore, PTH antagonized the angiotensin II-induced loss of cell function. Conclusion Low concentrations of PTH improve cell shortening by increasing calcium load at rest. By this mechanism cardiomyocytes compensate reduced extracellular calcium levels as they occur in patients with heart failure.

Calcitonin gene-related peptide (CGRP)-alpha is expressed in heart ventricles in sensory nerves and cardiomyocytes. It modifies inotropism and induces ischaemic preconditioning. This study investigates the effect of CGRP-alpha on the contractile responsiveness of isolated adult ventricular rat cardiomyocytes and the effect of chronic hypertension on this interaction. Cardiomyocytes were isolated and paced at 0.5-2.0 Hz. Cell shortening was recorded via a line camera with a reading frame of 500 Hz. CGRP-alpha exerted a dual effect on cardiomyocytes with a positive contractile effect at 10nM and a negative contractile effect at 10 pM. CGRP-alpha(8-37), a calcitonin receptor-like receptor (CRLR) antagonist, attenuated the positive contractile effect. H89, a protein kinase A antagonist, converted the positive contractile effect into a negative contractile effect. The negative contractile effect was converted again back to a positive contractile effect in the presence of l-nitro arginine. In cardiomyocytes isolated from spontaneously hypertensive rats (SHR) the mRNA expression of CRLR and the receptor-associated modifier protein (RAMP)-2 were lower. However, on the protein level CLRL was up-regulated, RAMP2 expression remained unchanged, and eNOS expression was down-regulated in these cells. These cells responded with a pure positive contractile response. In Langendorff preparations, CGRP-alpha slightly reduced the rate pressure product in hearts from normotensive rats but it caused an increase in hearts from SHR. In conclusion, it is shown that CGRP-alpha exerts dual effects on cardiomyocytes favouring the negative contractile effect at very low concentrations. This effect is compensated in chronic pressure-overloaded hearts and converted into a positive inotropism.

The activation of NO/cGMP pathways can induce pro-apoptotic pathways in cardiomyocytes although only a small number of cardiomyocytes fulfill the criteria of apoptosis. The same pathways reduce the contractile performance of cardiomyocytes. In the present study, we tested the hypothesis that exposure of cells to NO/cGMP for 24 h decrease their contractile performance due to an activation of pro-apoptotic pathways. Experiments were performed on freshly isolated and cultured adult ventricular rat cardiomyocytes. Cells were incubated with 8-bromo-cyclo-GMP (100 nmol/L-1 micromol/L), the NO donor SNAP (1 nmol/L-100 micromol/L), or the guanylyl cyclase activator YC-1 (3 micromol/L). Cell shortening, contraction and relaxation velocities, and diastolic cell lengths were determined at beating frequencies of 0.5, 1, and 2 Hz 24 h later. The activation of pro-apoptotic pathways was determined by staining of cardiomyocytes with an antibody directed against active caspase-3 and quantification of the number of apoptotic cells (annexin staining). Caspase-3 activation and an increase in the number of apoptotic cells was observed, but only at the highest concentrations tested (8-bromo-cyclo-GMP: 1-10 mmol/L; SNAP: 1-100 micromol/L). At these concentrations, none of the drugs decreased the mean cell shortening of cardiomyocytes. However, at concentrations lower than those required for induction of apoptotic cell death, the diastolic cell lengths and sarcomere lengths increased but cell shortening decreased. In conclusion, low concentrations of either NO or cGMP cause a desensitization of myofibrils, as indicated by elongated cell shapes, increased sarcomere lengths and reduced load-free cell shortening. High concentrations of NO/cGMP induce caspase-3 activation and increase the number of cells fulfilling the criteria of apoptotic cell death but did not impair cell function. Therefore, induction of apoptotic cell death per se seems not to contribute to the loss of contractile efficiency on the cellular level.

Objective: Myocardial ischemia has been shown to induce apoptosis of endothelial cells (EC). However, the mechanism of this endothelial injury is still poorly understood. To analyse the signaling pathway of ischemia-induced EC apoptosis was the aim of the present study. Methods: The primary culture of rat coronary EC was exposed to simulated ischemia (glucose-free anoxia at pHo 6.4). Apoptosis was defined by staining of nuclei with Hoechst-33342 and TUNEL. Cytosolic Ca2+ and pH were measured with Fura-2 and BCECF, respectively. Results: Apoptosis (29.2±1.7% of cells) induced by exposure to simulated ischemia for 2 h was accompanied by cytosolic Ca2+ overload (1090±52 nmol/l) and acidosis (pHi=6.52±0.13). Simulated ischemia had no significant effect on caspase-8 cleavage, but induced cleavage of caspase-3 and caspase-12 and led to a slight release of cytochrome C . Prevention of cytosolic acidosis (anoxia at pHo 7.4) had no effect on cytochrome C release, but significantly reduced apoptosis, attenuated cytosolic Ca2+ overload, and prevented cleavage of caspase-12. A similar effect was achieved by inhibition of Ca2+ release channels in the endoplasmic reticulum with ryanodine and xestospongin C. Knock-down of caspase-12 with small interfering RNA suppressed caspase-3 activation and reduced apoptotic cell number by about 70%. Conclusion: Acidosis, rather than anoxia, is an important trigger of apoptosis in EC under simulated ischemia. The main pathway of the simulated ischemia-induced apoptosis consists of the Ca2+ leak from the ER followed by activation of caspase-12 and caspase-3.

Parathyroid hormone-related peptide (PTHrP) is known to be a strong vasorelaxant peptide. The mechanisms by which PTHrP reduces the coronary resistance of the rat heart have not been worked out but seem to be independent of the classical PTH/PTHrP receptor-mediated, cAMP-dependent effect. In this study we hypothesized that PTHrP reduces the coronary resistance of the rat heart via endothelial cell hyperpolarization. Isolated microvascular endothelial cells from rat heart were incubated with PTHrP(1-36), and changes in the membrane potential were recorded via DiBAC fluorescence. Cells exposed to PTHrP showed a hyperpolarization of approximately 7mV. In the isolated Langendorff preparation, PTHrP-dependent vasodilatation of l-nitro-arginine-exposed hearts was abolished under depolarizing conditions (high potassium). Denudation of the endothelial cell layer significantly impaired the vasodilatory effect of PTHrP. In the presence of H89 (a cAMP/protein kinase A pathway antagonist) and indomethacin (a cyclooxygenase inhibitor), PTHrP dilated the vessels. In conclusion, PTHrP exerted a nitric oxide-independent vasodilatory effect that depends on endothelial cell hyperpolarization.

Objective Experimental and clinical studies have shown that administration of insulin during reperfusion is cardioprotective, but the underlying mechanisms are still unknown. In this study, we investigated in isolated rat cardiomyocytes subjected to hypoxia and reoxygenation whether administration of insulin during reoxygenation reduces reoxygenation-induced hypercontracture, a hallmark of acute reperfusion injury. The effects of insulin on potential pro-survival kinases, i.e., PI 3-kinase, NO synthase (eNOS), and cGMP-dependent protein kinase (PKG), and on cytosolic Ca2+ control in reoxygenated cardiomyocytes were investigated. Results Administration of insulin (10mU/L) during reoxygenation protected cardiomyocytes against hypercontracture development (cell length as % of end-hypoxic length: control 61.6±3.2; insulin 76.3±2.9*; n =26; * p

Objective Angiotensin II stimulation increases the formation of reactive oxygen species (ROS), the phosphorylation of p38 mitogen-activated protein kinase (MAPK), and the expression of transforming growth factor beta (TGFβ) in adult cardiomyocytes. The aim of this study was to determine the involvement of PI 3-kinase and to specify the participation of different isoforms in the angiotensin II-induced formation of ROS in comparison to the hypertrophic pathway triggered by α-adrenoceptor stimulation. Methods Freshly isolated myocytes were used to examine formation of ROS via H2DCF fluorescence. p38 MAPK phosphorylation, p70S6-kinase phosphorylation, PI 3-kinase, and TGFβ expression were measured by Western blotting. Sense and antisense oligonucleotides were used to down-regulate diverse PI 3-kinase isoforms. Hypertrophy was measured by 14C-phenylalanine incorporation and cell volume. Results Inhibition of PI 3-kinase by Ly294002 or wortmannin, two inhibitors, decreased formation of ROS, phosphorylation of p38 MAPK, and TGFβ expression. Down-regulation of the p110β isoform by antisense oligonucleotides inhibited the angiotensin II-induced signalling pathway but not the α-adrenoceptor-mediated hypertrophic growth of cardiomyocytes. In contrast, down-regulation of the p110α isoform decreased the α-adrenoceptor-mediated hypertrophic growth of cardiomyocytes but did not affect the angiotensin II-mediated signalling pathway. Conclusion Thus, our study identifies an involvement of PI 3-kinase in the angiotensin II-induced formation of ROS and provides a biochemical basis for ligand-specific responses for angiotensin II and α-adrenoceptor stimulation as relates to hypertrophy.

Objective: Ischemia–reperfusion provokes barrier failure of the coronary microvasculature, leading to myocardial edema development that jeopardizes functional recovery of the heart during reperfusion. Here, we tested whether adenosine 5′-triphosphate (ATP), either exogenously applied or spontaneously released during reperfusion, protects the endothelial barrier against an imminent reperfusion injury and whether interventions preventing ATP breakdown augment this protective ATP effect. Methods Cultured microvascular coronary endothelial monolayers and isolated-perfused hearts of rat were used. Results: After ischemic conditions were induced, reperfusion of endothelial monolayers activated the endothelial contractile machinery and caused intercellular gap formation. It also led to the release of ATP. When its breakdown was inhibited by 6- N , N -diethyl-β,γ-dibromomethylene-d-ATP (ARL 67156; 100 μM), a selective ectonucleotidase inhibitor, contractile activation and gap formation were significantly reduced. Reperfusion in the presence of exogenously added ATP (10 μM) plus ARL caused an additional reduction of both aforementioned effects. In contrast, elevation of ATP degradation by apyrase (1 U/ml), a soluble ectonucleotidase, or addition of adenosine (10 μM) provoked an increase in gap formation during reperfusion that could be completely inhibited by 8-phenyltheophylline (8-PT; 10 μM), an adenosine receptor antagonist. In Langendorff-perfused rat hearts, the reperfusion-induced increase in water content was significantly reduced by ARL plus ATP. Under conditions favouring ATP degradation, an increase in myocardial edema was observed that could be blocked by 8-PT. Conclusion ATP, either released from cells or exogenously applied, protects against reperfusion-induced failure of the coronary endothelial barrier. Inhibition of ATP degradation enhances the stabilizing effect of ATP on barrier function.

Catecholamines seem to play a major role in the initial response of the heart to pressure overload. The mechanisms by which alpha(1A)-adrenoceptor stimulation increases protein synthesis and subsequently cell size have been worked out in the past. However, little is known about the functional consequence of this type of hypertrophy. Recent transgenic work seems to indicate an adaptive character of this response, but mechanistic insights have yet to be established. The present study investigates whether chronic (overnight) exposure of cardiomyocytes to phenylephrine, an alpha-adrenoceptor agonist, modifies the expression of calcium-handling proteins and identifies key elements of signal transduction pathways leading to such alterations. Cardiomyocytes exposed to phenylephrine had elevated expression of SR-calcium ATPase (SERCA), but not of the sodium-calcium exchanger (NCX). SERCA induction persisted in the presence of protein kinase C (PKC) inhibitors, but required an increase in diastolic cell calcium levels via activation of the sodium-proton exchanger (NHE) and the reverse mode of the NCX. Downstream of an increase in resting cell calcium concentrations an activation of the calcineurin/NFAT pathway was found to be responsible for SERCA2 induction. Transfection of cardiomyocytes with decoys directed against NFAT activity inhibited the increase in SERCA2 expression. Decoys did not inhibit the concomitant PKC-dependent increase in hypertrophic growth. In the absence of SERCA up-regulation, hypertrophied cardiomyocytes were unable to maintain normal, load-free cell shortening. In conclusion, our data give mechanistic insights into the adaptional process during alpha-adrenoceptor-dependent myocardial hypertrophy.

Objective : Reperfusion injury of the myocardium is characterised by development of cardiomyocyte hypercontracture. Previous studies have shown that cGMP-mediated stimuli protect against reperfusion injury, but the cellular mechanism is still unknown. Methods : To simulate ischemia/reperfusion, adult rat cardiomyocytes were incubated anoxically (pHo 6.4) and then reoxygenated (pHo 7.4). Cytosolic calcium [Ca2+] i (fura-2 ratio), pH i (BCECF ratio), cell length, and phospholamban phosphorylation were analysed. Under simulated ischemia cardiomyocytes develop [Ca2+] i overload. When reoxygenated they rapidly undergo hypercontracture, triggered by oscillations of [Ca2+] i . We investigated whether cGMP-mediated stimuli can modulate [Ca2+] i or pH i recovery and whether this contributes to their protective effect. Membrane-permeable cGMP analogues, 8-bromo-cGMP (1 mmol/L) or 8-pCPT-cGMP (10 μmmol/L), or a receptor-mediated activator of particulate guanylyl cyclase, urodilatin (1 μmol/L), were applied. Results : The investigated stimuli protect against reoxygenation-induced hypercontracture (cell length as percent of end-ischemic length; control: 68 ± 1.6; 8-bromo-cGMP: 88 ± 1.5*; 8-pCPT-cGMP: 84 ± 2.9*; urodilatin: 87 ± 1.1*; n =24; * p

The hypothesis tested was that reoxygenation-induced contracture of myocardial cells, a form of reperfusion injury, can be due to a rigor-type mechanism. Isolated adult cardiomyocytes were exposed to 30- or 60-min anoxia (pH 6.4) and reoxygenation (pH 7.4). In cardiomyocytes, cytosolic Ca(2+) and cell length, and in isolated rat hearts left ventricular end-diastolic pressure (LVEDP) were measured. During reoxygenation, cardiomyocytes developed contracture. When energy recovery was slowed down, less Ca(2+) overload was required for contracture: (1) after 30-min anoxia Ca(20) (cytosolic Ca(2+) concentration in cells with 20% cell length reduction) was 1.42 +/- 0.11 micromol/l; (2) after 30-min anoxia with partial mitochondrial inhibition during reoxygenation (NaCN, 0.1 mmol/l) Ca(20) was reduced to 0.69 +/- 0.05 micromol/l; (3) after 60-min anoxia Ca(20) was reduced to 0.78 +/- 0.05 micromol/l and (4) when energy recovery was accelerated (succinate, 0.2 mmol/l), Ca(20) rose to 1.35 +/- 0.05 micromol/l. In isolated hearts, the reperfusion-induced rise in LVEDP was modulated by the same interventions. Slow recovery of energy production favors reoxygenation-induced contracture in cardiomyocytes and hearts. This shows that rigor contracture contributes to reoxygenation-induced cell injury.

Objective: Barrier function of coronary endothelium becomes disturbed by ischemia–reperfusion. We investigated the mechanism of reperfusion-induced endothelial gap formation in monolayers of cultured endothelial cells (CEC) of the rat, exposed to simulated ischemia (40 min anoxia, pHo 6.4) and reperfusion (30 min reoxygenation, pHo 7.4). Methods: Cytosolic Ca2+ (fura-2) and intercellular gap formation (planimetrical analysis) were determined. Reoxygenation conditions were varied: (a) continuing perfusion at pHo 6.4, (b) with or without glucose (2.5 mM), (c) in presence of NaCN (2 mM), (d) with Ca2+ (10 mM) or BAPTA/AM (25 μM), (e) in the presence of myosin light chain kinase inhibitors ML-7 (5 μM) or wortmannin (1 μM). Results: During anoxia, CEC developed cytosolic Ca2+ overload which was not reversed during 30 min reoxygenation. Intercellular gap formation started during anoxia, but was increased during reoxygenation. Reoxygenation-related gap formation was largest in presence of glucose, lower when glucose was withdrawn or NaCN was added. Presence of ML-7 or wortmannin also reduced gap formation during reoxygenation. Conclusions: Reoxygenation induces gap formation. This is dependent on (i) Ca2+ overload during reoxygenation, (ii) energy production and (iii) activation of myosin light chain kinase. Together these results indicate that activation of the endothelial contractile machinery is the underlying cause.