NOS1 induces NADPH oxidases and impairs contraction kinetics in aged murine ventricular myocytes.

Nitric oxide (NO) modulates calcium transients and contraction of cardiomyocytes. However, it is largely unknown whether NO contributes also to alterations in the contractile function of cardiomyocytes during aging. Therefore, we analyzed the putative role of nitric oxide synthases and NO for the age-related alterations of cardiomyocyte contraction. We used C57BL/6 mice, nitric oxide synthase 1 (NOS1)-deficient mice (NOS1(-/-)) and mice with cardiomyocyte-specific NOS1-overexpression to analyze contractions, calcium transients (Indo-1 fluorescence), acto-myosin ATPase activity (malachite green assay), NADPH oxidase activity (lucigenin chemiluminescence) of isolated ventricular myocytes and cardiac gene expression (Western blots, qPCR). In C57BL/6 mice, cardiac expression of NOS1 was upregulated by aging. Since we found a negative regulation of NOS1 expression by cAMP in isolated cardiomyocytes, we suggest that reduced efficacy of ß-adrenergic signaling that is evident in aged hearts promotes upregulation of NOS1. Shortening and relengthening of cardiomyocytes from aged C57BL/6 mice were decelerated, but were normalized by pharmacological inhibition of NOS1/NO. Cardiomyocytes from NOS1(-/-) mice displayed no age-related changes in contraction, calcium transients or acto-myosin ATPase activity. Aging increased cardiac expression of NADPH oxidase subunits NOX2 and NOX4 in C57BL/6 mice, but not in NOS1(-/-) mice. Similarly, cardiac expression of NOX2 and NOX4 was upregulated in a murine model with cardiomyocyte-specific overexpression of NOS1. We conclude that age-dependently upregulated NOS1, putatively via reduced efficacy of ß-adrenergic signaling, induces NADPH oxidases. By increasing nitrosative and oxidative stress, both enzyme systems act synergistically to decelerate contraction of aged cardiomyocytes.

Direct inhibition, but indirect sensitization of pacemaker activity to sympathetic tone by the interaction of endotoxin with HCN-channels.

In critically ill patients regulation of heart-rate is often severely disturbed. Interaction of bacterial endotoxin (lipopolysaccharide, LPS) with hyperpolarization-activated cyclic nucleotide-gated cation-(HCN)-channels may interfere with heart-rate regulation. This study analyzes the effect of LPS, the HCN-channel blocker ivabradine or Ca(2+) -channel blockers (nifedipine, verapamil) on pacemaking in spontaneously beating neonatal rat cardiomyocytes (CM) in vitro. In vivo, the effect of LPS on the heart-rate of adult CD1-mice with and without autonomic blockade is analyzed telemetrically. LPS (100 ng/mL) and ivabradine (5 µg/mL) reduced the beating-rate of CM by 20.1% and 24.6%, respectively. Coincubation of CM with both, LPS and ivabradine, did not further reduce the beating-rate, indicating interaction of both compounds with HCN-channels, while coincubation with Ca(2+) -channel blockers and LPS caused additive beating-rate reduction. In CD1-mice (containing an active autonomic-nervous-system), injection of LPS (0.4 mg/kg) expectedly resulted in increased heart-rate. However, if the autonomic nervous system was blocked by propranolol and atropine, in line with the in vitro data, LPS induced a significant reduction of heart-rate, which was not additive to ivabradine. The in vivo and in vitro results indicate that LPS interacts with HCN-channels of cardiomyocytes. Thus, LPS indirectly sensitizes HCN-channels for sympathetic activation (tachycardic-effect), and in parallel directly inhibits channel activity (bradycardic-effect). Both effects may contribute to the detrimental effects of septic cardiomyopathy and septic autonomic dysfunction.

Inhibition of cardiac pacemaker channel hHCN2 depends on intercalation of lipopolysaccharide into channel-containing membrane microdomains.

Depressed heart rate variability in severe inflammatory diseases can be partially explained by the lipopolysaccharide (LPS)-dependent modulation of cardiac pacemaker channels. Recently, we showed that LPS inhibits pacemaker current in sinoatrial node cells and in HEK293 cells expressing cloned pacemaker channels, respectively. The present study was designed to verify whether this inhibition involves LPS-dependent intracellular signalling and to identify structures of LPS responsible for pacemaker current modulation. We examined the effect of LPS on the activity of human hyperpolarization-activated cyclic nucleotide-gated channel 2 (hHCN2) stably expressed in HEK293 cells. In whole-cell recordings, bath application of LPS decreased pacemaker current (IhHCN2) amplitude. The same protocol had no effect on channel activity in cell-attached patch recordings, in which channels are protected from the LPS-containing bath solution. This demonstrates that LPS must interact directly with or close to the channel protein. After cleavage of LPS into lipid A and the polysaccharide chain, neither of them alone impaired IhHCN2, which suggests that modulation of channel activity critically depends on the integrity of the entire LPS molecule. We furthermore showed that ß-cyclodextrin interfered with LPS-dependent channel modulation predominantly via scavenging of lipid A, thereby abrogating the capability of LPS to intercalate into target cell membranes. We conclude that LPS impairs IhHCN2 by a local mechanism that is restricted to the vicinity of the channels. Furthermore, intercalation of lipid A into target cell membranes is a prerequisite for the inhibition that is suggested to depend on the direct interaction of the LPS polysaccharide chain with cardiac pacemaker channels.

Differential reduction of HCN channel activity by various types of lipopolysaccharide.

Recently it was shown that lipopolysaccharide (LPS) impairs the pacemaker current in human atrial myocytes. It was speculated that reduced heart rate variability (HRV), typical of patients with severe sepsis, may partially be explained by this impairment. We evaluated the effect of various types of LPS on the activity of human hyperpolarization-activated cyclic nucleotide-gated channel 2 (hHCN2) expressed in HEK293 cells, and on pacemaker channels in native murine sino-atrial node (SAN) cells, in order to determine the structure of LPS necessary to modulate pacemaker channel function. Application of LPS caused a robust inhibition of hHCN2-mediated current (I(hHCN2)) owing to a negative shift of the voltage dependence of current activation and to a reduced maximal conductance. In addition, kinetics of channel gating were modulated by LPS. Pro-inflammatory LPS-types lacking the O-chain did not reduce I(hHCN2), whereas pro-inflammatory LPS-types containing the O-chain reduced I(hHCN2). On the other hand, a detoxified LPS without inflammatory activity, but containing the O-chain reduced I(hHCN2). Similar observations were made in HEK293 cells expressing hHCN4 and in murine SAN cells. This mechanistic analysis showed the novel finding that the O-chain of LPS is required for reduction of HCN channel activity. In the clinical situation the observed modulation of HCN channels may slow down diastolic depolarization of pacemaker cells and, hence, influence heart rate variability and heart rate.

Cardiac overexpression of the human 5-HT4 receptor in mice.

Serotonin (5-HT) exerts pleiotropic effects in the human cardiovascular system. Some of the effects are thought to be mediated via 5-HT(4) receptors, which are expressed in the human atrium and in ventricular tissue. However, a true animal model to study these receptors in more detail has been hitherto lacking. Therefore, we generated, for the first time, a transgenic (TG) mouse with cardiac myocyte-specific expression of the human 5-HT(4) receptor. RT-PCR and immunohistochemistry revealed expression of the receptor at the mRNA and protein levels. Stimulation of isolated cardiac preparations by isoproterenol increased phospholamban phosphorylation at Ser(16) and Thr(17) sites. 5-HT increased phosphorylation only in TG mice but not in wild-type (WT) mice. Furthermore, 5-HT increased contractility in isolated perfused hearts from TG mice but not WT mice. These effects of 5-HT could be blocked by the 5-HT(4) receptor-selective antagonist GR-125487. An intravenous infusion of 5-HT increased left ventricular contractility in TG mice but not in WT mice. Similarly, the increase in contractility by 5-HT in isolated cardiomyocytes from TG mice was accompanied by and probably mediated through an increase in L-type Ca(2+) channel current and in Ca(2+) transients. In intact animals, echocardiography revealed an inotropic and chronotropic effect of subcutaneously injected 5-HT in TG mice but not in WT mice. In isolated hearts from TG mice, spontaneous polymorphic atrial arrhythmias were noted. These findings demonstrate the functional expression of 5-HT(4) receptors in the heart of TG mice, and a potential proarrhythmic effect in the atrium. Therefore, 5-HT(4) receptor-expressing mice might be a useful model to mimic the human heart, where 5-HT(4) receptors are present and functional in the atrium and ventricle of the healthy and failing heart, and to investigate the influence of 5-HT in the development of cardiac arrhythmias and heart failure.

Reduction of insulin secretion in the insulinoma cell line INS-1 by overexpression of a Ca(v)2.3 (alpha1E) calcium channel antisense cassette.

OBJECTIVE: Multiple types of voltage-activated Ca(2+) channels (T, L, N, P, Q and R type) coordinate a variety of Ca(2+)-dependent processes in neurons and neuroendocrine cells. In insulinoma cell lines as well as in endocrine tissues, the non-L-type alpha1E (Ca(v)2.3) subunit is expressed as the tissue-specific splice variant alpha1Ee. DESIGN AND METHODS: To understand the functional role of alpha1E-containing Ca(2+) channels, antisense alpha1E mRNA was overexpressed in INS-1 cells by stable transfection of an antisense alpha1E cassette cDNA. As controls, either a sense alpha1E cassette or a control vector containing enhanced green fluorescent protein as an unrelated gene was stably transfected. The overexpression of each transfected cassette cDNA was recorded by RT-PCR. RESULTS: In three independent antisense alpha1E INS-1 clones, the glucose-induced insulin release was significantly reduced as compared with wild-type INS-1 cells and with a sense alpha1E INS-1 clone. However, in the antisense INS-1 clones, the KCl-induced insulin release was less impaired by overexpressing the antisense alpha1E cassette than the glucose-induced insulin release, leading to the assumption that glucose (15 mmol/l) and KCl (25 mmol/l) finally depolarize the membrane potential to a different extent. CONCLUSION: alpha1E is involved in glucose-induced insulin secretion probably by influencing the excitability of INS-1 cells.

NADPH oxidase-derived superoxide impairs calcium transients and contraction in aged murine ventricular myocytes.

Since aging increases oxidative stress, we analyzed the contribution of reactive oxygen species (ROS) to the contractile dysfunction of aged ventricular myocytes and investigated whether short-term interference with ROS formation could normalize contractile performance. Isolated ventricular myocytes from young (2-4 months) and aged (24-26 months) male mice (C57BL/6) were used. We analyzed sarcomere shortening and calcium transients (Indo-1 fluorescence) of voltage clamped ventricular myocytes and myofilament ATPase activity (malachite green assay). Expression of calcium handling proteins (Western blots) and NADPH oxidase subunits (real-time PCR) was quantified, as well as NADPH oxidase activity (lucigenin chemiluminescence). We found that aged myocytes showed decelerated shortening/relengthening without changes in fractional shortening. Calcium transient decay was similarly decelerated, but the amplitude of calcium transients was increased with aging. Calcium sensitivity of myofilaments of aged myocytes was reduced. These age-dependent changes occurred without altered calcium handling protein expression but were reversed by the superoxide scavenger tiron. Aged myocytes showed increased NADPH oxidase expression and activity. Pharmacological inhibition of NADPH oxidase (diphenylene iodonium; apocynin) normalized age-dependent deceleration of shortening/relengthening. In summary, we show that increased superoxide formation by upregulated NADPH oxidase contributes significantly to age-dependent alterations in calcium handling and contractility of murine ventricular myocytes.

Inotropic effects of L-lysine in the mammalian heart.

We studied the effects of L-lysine in cardiac preparations of mice and men. Of note, L-lysine increased force of contraction in a concentration- and time-dependent manner in isolated electrically paced left atrium of mouse and in human right atrium. It further increased heart rate and left ventricular pressure in the isolated perfused mouse heart. In isolated adult mouse cardiomyocytes, the contractility as assessed by edge detection was increased as well as the Ca(2+) transients after electrically pacing by field stimulation. However, using the patch clamp technique, no effect of L-lysine on action potential duration from a constant holding potential or on current through L-type calcium channels could be observed. However, L-lysine led to a depolarization of unclamped cells. Furthermore, effects of L-lysine were stereospecific, as they were not elicited by D-lysine. The inotropic effects of L-lysine were not abrogated by additionally applied L-ornithine or L-arginine (known inhibitors of lysine transport). However, L-lysine (5 mM) shifted the concentration-response curve for a positive inotropic effect of 5-hydroxytryptamine (5-HT; serotonin) in atrium of transgenic mice (with cardiac specific overexpression of 5-HT(4) receptors) to higher concentrations. In summary, we describe a novel positive inotropic effect of an essential amino acid, L-lysine, in the mammalian heart. One might speculate that L-lysine treatment under certain conditions could sustain cardiac performance. Moreover, L-lysine is able to block, at least in part, cardiac 5-HT(4) receptors.

The molecular chaperone hsp70 interacts with the cytosolic II-III loop of the Cav2.3 E-type voltage-gated Ca2+ channel.

Multiple types of voltage-activated Ca2+ channels (T, L, N, P, Q, R type) coexist in excitable cells and participate in synaptic differentiation, secretion, transmitter release, and neuronal plasticity. Ca2+ ions entering cells trigger these events through their interaction with the ion channel itself or through Ca2+ binding to target proteins initiating signalling cascades at cytosolic loops of the ion conducting subunit (Cava1). These loops interact with target proteins in a Ca2+-dependent or independent manner. In Cav2.3-containing channels the cytosolic linker between domains II and III confers a novel Ca2+ sensitivity to E-type Ca2+ channels including phorbol ester sensitive signalling via protein kinase C (PKC) in Cav2.3 transfected HEK-293 cells. To understand Ca2+ and phorbol ester mediated activation of Cav2.3 Ca2+ channels, protein interaction partners of the II-III loop were identified. FLAG-tagged II-III - loop of human Cav2.3 was over-expressed in HEK 293 cells, and the molecular chaperone hsp70, which is known to interact with PKC, was identified as a novel functional interaction partner. Immunopurified II-III loop-protein of neuronal and endocrine Cav2.3 splice variants stimulate autophosphorylation of PKCa, leading to the suggestion that hsp70--binding to the II-III loop--may act as an adaptor for Ca2+ dependent targeting of PKC to E-type Ca2+ channels.

Characterization of auto-regulation of the human cardiac alpha1 subunit of the L-type calcium channel: importance of the C-terminus.

The carboxyl terminal of the L-type calcium channel alpha1C subunit comprises approximately one third of the primary structure of the alpha1 subunit (> 700 amino acids residues). This region is sensitive to limited posttranslational processing. In heart and brain the alpha1C subunits are found to be truncated but the C-terminal domain remains functionally present. Based on our previous data we hypothesized that the distal C-terminus (approximately residues 1650-1950) harbors an important, predominantly inhibitory domain. We generated C-terminal-truncated alpha1C mutants, and after expressing them in combination with a beta3 subunit in HEK-293 cells, electrophysiological experiments were carried out. In order to dissect the important inhibitory part of the C-terminus, trypsin was dialyzed into the cells. The data provide evidence that there are multiple residues within the inhibitory domain that are crucial to the inhibitory process as well as to the enhancement of expressed current by intracellular application of proteases. In addition, the expression of the chimeric mutant alpha(1C)delta1673-DRK1 demonstrated that the C-terminal is specific for the heart channel.

Alternate splicing in the cytosolic II-III loop and the carboxy terminus of human E-type voltage-gated Ca(2+) channels: electrophysiological characterization of isoforms.

There is growing evidence that Ca(v)2.3 (alpha1E, E-type) transcripts may encode the ion-conducting subunit of a subclass of R-type Ca(2+) channels, a heterogeneous group of channels by definition resistant to blockers of L-, N-, and P/Q-type Ca(2+) channels. To understand whether splice variation of Ca(v)2.3 contributes to the divergence of R-type channels, individual variants of Ca(v)2.3 were constructed and expressed in HEK-293 cells. With Ba(2+) as charge carrier, the tested biophysical properties were similar. In Ca(2+), the inactivation time course was slower and the recovery from short-term inactivation was faster; however, this occurred only in variants containing a 19-amino-acid-long insertion, which is typical for neuronal Ca(v)2.3 Ca(2+) channel subunits. This different Ca(2+) sensitivity is not responsible for the major differences between various R-type channels, and future studies might clarify its importance for in vivo synaptic or dendritic integration and the reasons for its loss in endocrine Ca(v)2.3 splice variants.

The cytosolic II-III loop of Cav2.3 provides an essential determinant for the phorbol ester-mediated stimulation of E-type Ca2+ channel activity.

There is growing evidence that E-type voltage dependent Ca(2+) channels (Ca(v)2.3) are involved in triggering and controlling pivotal cellular processes like neurosecretion and long-term potentiation. The mechanism underlying a novel Ca(2+) dependent stimulation of E-type Ca(2+) channels was investigated in the context of the recent finding that influx of Ca(2+) through other voltage dependent Ca(2+) channels is necessary and sufficient to directly activate protein kinase C (PKC). With Ba(2+) as charge carrier through Ca(v)2.3 channel alpha(1) subunits expressed in HEK-293 cells, activation of PKC by low concentrations of phorbol ester augmented peak I(Ba) by approximately 60%. In addition, the non-inactivating fraction of I(Ba) was increased by more than three-fold and recovery from short-term inactivation was accelerated. The effect of phorbol ester on I(Ba) was inhibited by application of the specific PKC inhibitor bisindolylmaleimide I. With Ca(2+) as charge carrier, application of phorbol ester did not change the activity of Ca(v)2.3 currents but they were modified by the PKC inhibitor bisindolylmaleimide I. These results suggest that with Ca(2+) as charge carrier the incoming Ca(2+) can activate PKC, thereby augmenting Ca(2+) influx into the cytosol. No modulation of Ca(v)2.3 channels by PKC was observed when an arginine rich region in the II-III loop of Ca(v)2.3 was eliminated. Receptor independent stimulation of PKC and its interaction with Ca(v)2.3 channels therefore represents an important positive feedback mechanism to decode electrical signals into a variety of cellular functions.

Ca2+-sensitive regulation of E-type Ca2+ channel activity depends on an arginine-rich region in the cytosolic II-III loop.

Ca2+-dependent regulation of L-type and P/Q-type Ca2+ channel activity is an important mechanism to control Ca2+ entry into excitable cells. Here we addressed the question whether the activity of E-type Ca2+ channels can also be controlled by Ca2+. Switching from Ba2+ to Ca2+ as charge carrier increased within 50 s, the density of currents observed in HEK-293 cells expressing a human Cav2.3d subunit and slowed down the inactivation kinetics. Furthermore, with Ca2+ as permeant ion, recovery from inactivation was accelerated, compared to the recovery process recorded under conditions where the accumulation of [Ca2+]i was prevented. In a Ba2+ containing bath solution the Ca2+-dependent changes of E-type channel activity could be induced by dialysing the cells with 1 micro m free [Ca2+]i suggesting that an elevation of [Ca2+]i is responsible for these effects. Deleting 19 amino acids in the intracellular II-III linker (exon 19) as part of an arginine-rich region, severely impairs the Ca2+ responsiveness of the expressed channels. Interestingly, deletion of an adjacent homologue arginine-rich region activates channel activity but now independently from [Ca2+]i. As a positive feedback-regulation of channel activity this novel activation mechanism might determine specific biological functions of E-type Ca2+ channels.