Omecamtiv mecarbil augments cardiomyocyte contractile activity both at resting and systolic Ca2+ levels.

AIMS: Heart failure with reduced ejection fraction (HFrEF) is a disease with high mortality and morbidity. Recent positive inotropic drug developments focused on cardiac myofilaments, that is, direct activators of the myosin molecule and Ca2+ sensitizers for patients with advanced HFrEF. Omecamtiv mecarbil (OM) is the first direct myosin activator with promising results in clinical studies. Here, we aimed to elucidate the cellular mechanisms of the positive inotropic effect of OM in a comparative in vitro investigation where Ca2+ -sensitizing positive inotropic agents with distinct mechanisms of action [EMD 53998 (EMD), which also docks on the myosin molecule, and levosimendan (Levo), which binds to troponin C] were included. METHODS: Enzymatically isolated canine cardiomyocytes with intact cell membranes were loaded with Fura-2AM, a Ca2+ -sensitive, ratiometric, fluorescent dye. Changes in sarcomere length (SL) and intracellular Ca2+ concentration were recorded in parallel at room temperature, whereas cardiomyocyte contractions were evoked by field stimulation at 0.1 Hz in the presence of different OM, EMD, or Levo concentrations. RESULTS: SL was reduced by about 23% or 9% in the presence of 1 µM OM or 1 µM EMD in the absence of electrical stimulation, whereas 1 µM Levo had no effect on resting SL. Fractional sarcomere shortening was increased by 1 µM EMD or 1 µM Levo to about 152%, but only to about 128% in the presence of 0.03 µM OM. At higher OM concentrations, no significant increase in fractional sarcomere shortening could be recorded. Contraction durations largely increased, whereas the kinetics of contractions and relaxations decreased with increasing OM concentrations. One-micromole EMD or 1 µM Levo had no effects on contraction durations. One-micromole Levo, but not 1 µM EMD, accelerated the kinetics of cardiomyocyte contractions and relaxations. Ca2+ transient amplitudes were unaffected by all treatments. CONCLUSIONS: Our data revealed major distinctions between the cellular effects of myofilament targeted agents (OM, EMD, or Levo) depending on their target proteins and binding sites, although they were compatible with the involvement of Ca2+ -sensitizing mechanisms for all three drugs. Significant part of the cardiotonic effect of OM relates to the prolongation of systolic contraction in combination with its Ca2+ -sensitizing effect.

Pharmacological Modulation and (Patho)Physiological Roles of TRPM4 Channel-Part 2: TRPM4 in Health and Disease.

Transient receptor potential melastatin 4 (TRPM4) is a unique member of the TRPM protein family and, similarly to TRPM5, is Ca2+ sensitive and permeable for monovalent but not divalent cations. It is widely expressed in many organs and is involved in several functions; it regulates membrane potential and Ca2+ homeostasis in both excitable and non-excitable cells. This part of the review discusses the currently available knowledge about the physiological and pathophysiological roles of TRPM4 in various tissues. These include the physiological functions of TRPM4 in the cells of the Langerhans islets of the pancreas, in various immune functions, in the regulation of vascular tone, in respiratory and other neuronal activities, in chemosensation, and in renal and cardiac physiology. TRPM4 contributes to pathological conditions such as overactive bladder, endothelial dysfunction, various types of malignant diseases and central nervous system conditions including stroke and injuries as well as in cardiac conditions such as arrhythmias, hypertrophy, and ischemia-reperfusion injuries. TRPM4 claims more and more attention and is likely to be the topic of research in the future.

Erector spinae catheter for post-thoracotomy pain control in a premature neonate.

Ensuring respiratory stability with early tracheal extubation and adequate pain control is challenging in premature neonates after thoracotomy. Continuous erector spinae plane (ESP) block, a relatively new truncal nerve block, has the potential to provide analgesia for thoracic surgeries while reducing opioid use. However, there have been only a few reports utilising this technique in infants, and none in preterm neonates. We present the perioperative pain management of a preterm neonate requiring thoracotomy. Epidural analgesia was deemed contraindicated due to coexisting coagulopathy; therefore, an ESP catheter was placed. The patient was extubated at the end of the surgery and had excellent pain control with rectal acetaminophen, chloroprocaine infusion via the ESP catheter and with minimal opioid requirement. Continuous ESP block may be safe and effective for postoperative pain management in coagulopathic premature neonates. Chloroprocaine is an effective local anaesthetic in the erector spinae compartment, which has not been previously reported.

Frequency-dependent effects of omecamtiv mecarbil on cell shortening of isolated canine ventricular cardiomyocytes.

Omecamtiv mecarbil (OM) is a myosin activator agent developed for the treatment of heart failure. OM was reported to increase left ventricular ejection fraction and systolic ejection time, but little is known about the effect of heart rate on the action of OM. The present study, therefore, was designed to investigate the effects of OM on unloaded cell shortening and intracellular Ca2+ ([Ca2+]i) transients as a function of the pacing frequency. Isolated cardiomyocytes were stimulated at various frequencies under steady-state conditions. Cell length was monitored by an optical edge detector and changes in [Ca2+]i were followed using the Ca2+-sensitive dye Fura-2. At the pacing frequency of 1 Hz, OM (1-10 µM) significantly decreased both diastolic and systolic cell length, however, fractional shortening was augmented only by 1 µM OM. Time to peak tension and time of 90% relaxation were progressively increased by OM. At the frequency of 2 Hz, diastolic cell length was reduced by 10 µM OM to a larger extent than systolic cell length, resulting in a significantly decreased fractional shortening under these conditions. OM had no effect on the parameters of the [Ca2+]i transient at any pacing frequency. The results suggest that supratherapeutic concentrations of OM may decrease rather than increase the force of cardiac contraction especially in tachycardic patients.

Metabolic responses of Rhodococcus erythropolis PR4 grown on diesel oil and various hydrocarbons.

Rhodococcus erythropolis PR4 is able to degrade diesel oil, normal-, iso- and cycloparaffins and aromatic compounds. The complete DNA content of the strain was previously sequenced and numerous oxygenase genes were identified. In order to identify the key elements participating in biodegradation of various hydrocarbons, we performed a comparative whole transcriptome analysis of cells grown on hexadecane, diesel oil and acetate. The transcriptomic data for the most prominent genes were validated by RT-qPCR. The expression of two genes coding for alkane-1-monooxygenase enzymes was highly upregulated in the presence of hydrocarbon substrates. The transcription of eight phylogenetically diverse cytochrome P450 (cyp) genes was upregulated in the presence of diesel oil. The transcript levels of various oxygenase genes were determined in cells grown in an artificial mixture, containing hexadecane, cycloparaffin and aromatic compounds and six cyp genes were induced by this hydrocarbon mixture. Five of them were not upregulated by linear and branched hydrocarbons. The expression of fatty acid synthase I genes was downregulated by hydrocarbon substrates, indicating the utilization of external alkanes for fatty acid synthesis. Moreover, the transcription of genes involved in siderophore synthesis, iron transport and exopolysaccharide biosynthesis was also upregulated, indicating their important role in hydrocarbon metabolism. Based on the results, complex metabolic response profiles were established for cells grown on various hydrocarbons. Our results represent a functional annotation of a rhodococcal genome, provide deeper insight into molecular events in diesel/hydrocarbon utilization and suggest novel target genes for environmental monitoring projects.

The heat shock factor A4A confers salt tolerance and is regulated by oxidative stress and the mitogen-activated protein kinases MPK3 and MPK6.

Heat shock factors (HSFs) are principal regulators of plant responses to several abiotic stresses. Here, we show that estradiol-dependent induction of HSFA4A confers enhanced tolerance to salt and oxidative agents, whereas inactivation of HSFA4A results in hypersensitivity to salt stress in Arabidopsis (Arabidopsis thaliana). Estradiol induction of HSFA4A in transgenic plants decreases, while the knockout hsfa4a mutation elevates hydrogen peroxide accumulation and lipid peroxidation. Overexpression of HSFA4A alters the transcription of a large set of genes regulated by oxidative stress. In yeast (Saccharomyces cerevisiae) two-hybrid and bimolecular fluorescence complementation assays, HSFA4A shows homomeric interaction, which is reduced by alanine replacement of three conserved cysteine residues. HSFA4A interacts with mitogen-activated protein kinases MPK3 and MPK6 in yeast and plant cells. MPK3 and MPK6 phosphorylate HSFA4A in vitro on three distinct sites, serine-309 being the major phosphorylation site. Activation of the MPK3 and MPK6 mitogen-activated protein kinase pathway led to the transcriptional activation of the HEAT SHOCK PROTEIN17.6A gene. In agreement that mutation of serine-309 to alanine strongly diminished phosphorylation of HSFA4A, it also strongly reduced the transcriptional activation of HEAT SHOCK PROTEIN17.6A. These data suggest that HSFA4A is a substrate of the MPK3/MPK6 signaling and that it regulates stress responses in Arabidopsis.

The long polar fimbriae operon and its flanking regions in bovine Escherichia coli O157:H43 and STEC O136:H12 strains.

Long polar fimbriae (Lpf) are intestinal adhesins and important virulence factors of pathogenic Escherichia coli strains. We cloned and sequenced the lpf2-1 operon (lpf2ABCD) and its flanking regions of an intimin- and Shiga toxin-negative atypical E. coli O157:H43 strain of bovine origin and also sequenced the lpf2-1 operon of six additional atypical O157 bovine E. coli strains of various serotypes. Nucleotide sequence comparison of these lpf operons showed sequence conservation as they contained only four polymorphic nucleotide positions. Investigation of these O157 strains as well as 13 E. coli Reference Collection (ECOR) strains carrying the lpf2-1 allele revealed high degree of sequence conservation in the lpf2 flanking regions. The lpf2-1 allele is also present in a bovine Shiga toxin-producing E. coli STEC O136:H12 strain, and in vitro adherence assays revealed that the absence of lpf2-1 in this strain did not affect its host cell-binding properties. Our data indicate that lpf2 loci are highly conserved in E. coli isolates; however, its role in adherence might be masked by other uncharacterized adhesins.

Effects of the PKC inhibitors chelerythrine and bisindolylmaleimide I (GF 109203X) on delayed rectifier K+ currents.

Protein kinase C (PKC) inhibitors are useful tools for studying PKC-dependent regulation of ion channels. For this purpose, high PKC specificity is a basic requirement excluding any direct interaction between the PKC inhibitor and the ion channel. In the present study, the effects of two frequently applied PKC inhibitors, chelerythine and bisindolylmaleimide I, were studied on the rapid and slow components of the delayed rectifier K(+) current (I(Kr) and I(Ks)) in canine ventricular cardiomyocytes and on the human ether-à-go-go-related gene (hERG) channels expressed in human embryonic kidney (HEK) cells. The whole cell version of the patch clamp technique was used in all experiments. Chelerythrine and bisindolylmaleimide I (both 1 µM) suppressed I(Kr) in canine ventricular cells. This inhibition developed rapidly, suggesting a direct drug-channel interaction. In HEK cells heterologously expressing hERG channels, chelerythrine and bisindolylmaleimide I blocked hERG current in a concentration-dependent manner, having EC(50) values of 0.11 ± 0.01 and 0.76 ± 0.04 µM, respectively. Both chelerythrine and bisindolylmaleimide I strongly modified gating kinetics of hERG--voltage dependence of activation was shifted towards more negative voltages and activation was accelerated. Deactivation was slowed by bisindolylmaleimide I but not by chelerythrine. I(Ks) was not significantly altered by bisindolylmaleimide I and chelerythrine. No significant effect of 0.1 µM bisindolylmaleimide I or 0.1 µM PMA (PKC activator) was observed on I(Kr) arguing against significant contribution of PKC to regulation of I(Kr). It is concluded that neither chelerythrine nor bisindolylmaleimide I is suitable for selective PKC blockade due to their direct blocking actions on the hERG channel.

Ghrelin controls hippocampal spine synapse density and memory performance.

The gut hormone and neuropeptide ghrelin affects energy balance and growth hormone release through hypothalamic action that involves synaptic plasticity in the melanocortin system. Ghrelin binding is also present in other brain areas, including the telencephalon, where its function remains elusive. Here we report that circulating ghrelin enters the hippocampus and binds to neurons of the hippocampal formation, where it promotes dendritic spine synapse formation and generation of long-term potentiation. These ghrelin-induced synaptic changes are paralleled by enhanced spatial learning and memory. Targeted disruption of the gene that encodes ghrelin resulted in decreased numbers of spine synapses in the CA1 region and impaired performance of mice in behavioral memory testing, both of which were rapidly reversed by ghrelin administration. Our observations reveal an endogenous function of ghrelin that links metabolic control with higher brain functions and suggest novel therapeutic strategies to enhance learning and memory processes.

Direct visual and circadian pathways target neuroendocrine cells in primates.

The effect of light on neuroendocrine functions is thought to be mediated through retinal inputs to the circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN). The present studies were conducted to provide experimental evidence for this signaling modality in non-human primates. In the St. Kitts vervet monkey, anterograde tracing of SCN efferents revealed a monosynaptic pathway between the circadian clock and hypothalamic neurons producing luteinizing hormone-releasing hormone (LHRH). Using a variety of tracing techniques, direct retinal input was found to be abundant in the SCN and in other hypothalamic sites. Strikingly, in hypothalamic areas other than the SCN, primary visual afferents established direct contacts with neuroendocrine cells including those producing LHRH and dopamine, neurons that are the hypothalamic regulators of pituitary gonadotrops and prolactin. Thus, our data reveal for the first time in primates that light stimuli can reach the hypothalamo-pituitary-gonadal axis, directly providing a pathway independent of but parallel to that of the circadian clock for the photic modulation of hormone release.

Fasting activates the nonhuman primate hypocretin (orexin) system and its postsynaptic targets.

In rodents, hypocretin (HCRT, also called orexin) influences a variety of endocrine, autonomic, and metabolic functions. The present study was undertaken to determine whether the HCRT-producing circuit is involved in the hypothalamic regulation of homeostasis in primates as well. We studied female monkeys (Cercopithecus aethiops) that were either fed or fasted for 24 h. Immunocytochemistry revealed HCRT-producing perikarya exclusively in the lateral hypothalamus-perifornical region and dorsomedial hypothalamus of the monkey brain. HCRT axons and axon terminals were present in different parts of the hypothalamus and adjacent forebrain and thalamic nuclei. The 24-h fast resulted in an approximately 50% decline in circulating leptin levels and significantly elevated c-fos expression in the perifornical region; in the dorsomedial, ventromedial, and arcuate nuclei; and in the medial preoptic area. In the dorsomedial nucleus and perifornical region of fasted monkeys, three times more HCRT-neurons expressed nuclear c-fos than those of the normally fed controls. Neurons in different parts of the hypothalamus and basal forebrain that expressed c-fos, but did not contain HCRT, were targets of HCRT-immunopositive boutons establishing asymmetric synapses. In the arcuate nucleus, subsets of these HCRT-targeted c-fos-expressing cells contained neuropeptide Y. The present study provides the first experimental evidence to implicate HCRT in the hypothalamic regulation of homeostasis in primates. The fact that these lateral hypothalamic cells have leptin receptors and can be activated by a metabolic challenge and that they innervate diverse brain regions indicates that the HCRT system may be a key integrator of environmental cues in their regulation of diverse brain activity.