Natriuretic Peptide Response and Outcomes in Chronic Heart Failure With Reduced Ejection Fraction.

BACKGROUND: The GUIDE-IT (GUIDing Evidence Based Therapy Using Biomarker Intensified Treatment in Heart Failure) trial demonstrated that a strategy to "guide" application of guideline-directed medical therapy (GDMT) by reducing amino-terminal pro-B-type natriuretic peptide (NT-proBNP) was not superior to GDMT alone. OBJECTIVES: The purpose of this study was to examine the prognostic meaning of NT-proBNP changes following heart failure (HF) therapy intensification relative to the goal NT-proBNP value of 1,000 pg/ml explored in the GUIDE-IT trial. METHODS: A total of 638 study participants were included who were alive and had available NT-proBNP results 90 days after randomization. Rates of subsequent cardiovascular (CV) death/HF hospitalization or all-cause mortality during follow-up and Kansas City Cardiomyopathy Questionnaire (KCCQ) overall scores were analyzed. RESULTS: A total of 198 (31.0%) subjects had an NT-proBNP ≤1,000 pg/ml at 90 days with no difference in achievement of NT-proBNP goal between the biomarker-guided and usual care arms. NT-proBNP ≤1,000 pg/ml by 90 days was associated with longer freedom from CV/HF hospitalization or all-cause mortality (p < 0.001 for both) and lower adjusted hazard of subsequent HF hospitalization/CV death (hazard ratio: 0.26; 95% confidence interval: 0.15 to 0.46; p < 0.001) and all-cause mortality (hazard ratio: 0.34; 95% confidence interval: 0.15 to 0.77; p = 0.009). Regardless of elevated baseline concentration, an NT-proBNP ≤1,000 pg/ml at 90 days was associated with better outcomes and significantly better KCCQ overall scores (p = 0.02). CONCLUSIONS: Patients with heart failure with reduced ejection fraction whose NT-proBNP levels decreased to ≤1,000 pg/ml during GDMT had better outcomes. These findings may help to understand the results of the GUIDE-IT trial. (Guiding Evidence Based Therapy Using Biomarker Intensified Treatment [GUIDE-IT]; NCT01685840).

Effect of Natriuretic Peptide-Guided Therapy on Hospitalization or Cardiovascular Mortality in High-Risk Patients With Heart Failure and Reduced Ejection Fraction: A Randomized Clinical Trial.

Importance: The natriuretic peptides are biochemical markers of heart failure (HF) severity and predictors of adverse outcomes. Smaller studies have evaluated adjusting HF therapy based on natriuretic peptide levels ("guided therapy") with inconsistent results. Objective: To determine whether an amino-terminal pro-B-type natriuretic peptide (NT-proBNP)-guided treatment strategy improves clinical outcomes vs usual care in high-risk patients with HF and reduced ejection fraction (HFrEF). Design, Settings, and Participants: The Guiding Evidence Based Therapy Using Biomarker Intensified Treatment in Heart Failure (GUIDE-IT) study was a randomized multicenter clinical trial conducted between January 16, 2013, and September 20, 2016, at 45 clinical sites in the United States and Canada. This study planned to randomize 1100 patients with HFrEF (ejection fraction ≤40%), elevated natriuretic peptide levels within the prior 30 days, and a history of a prior HF event (HF hospitalization or equivalent) to either an NT-proBNP-guided strategy or usual care. Interventions: Patients were randomized to either an NT-proBNP-guided strategy or usual care. Patients randomized to the guided strategy (n = 446) had HF therapy titrated with the goal of achieving a target NT-proBNP of less than 1000 pg/mL. Patients randomized to usual care (n = 448) had HF care in accordance with published guidelines, with emphasis on titration of proven neurohormonal therapies for HF. Serial measurement of NT-proBNP testing was discouraged in the usual care group. Main Outcomes and Measures: The primary end point was the composite of time-to-first HF hospitalization or cardiovascular mortality. Prespecified secondary end points included all-cause mortality, total hospitalizations for HF, days alive and not hospitalized for cardiovascular reasons, the individual components on the primary end point, and adverse events. Results: The data and safety monitoring board recommended stopping the study for futility when 894 (median age, 63 years; 286 [32%] women) of the planned 1100 patients had been enrolled with follow-up for a median of 15 months. The primary end point occurred in 164 patients (37%) in the biomarker-guided group and 164 patients (37%) in the usual care group (adjusted hazard ratio [HR], 0.98; 95% CI, 0.79-1.22; P = .88). Cardiovascular mortality was 12% (n = 53) in the biomarker-guided group and 13% (n = 57) in the usual care group (HR, 0.94; 95% CI; 0.65-1.37; P = .75). None of the secondary end points nor the decreases in the NT-proBNP levels achieved differed significantly between groups. Conclusions and Relevance: In high-risk patients with HFrEF, a strategy of NT-proBNP-guided therapy was not more effective than a usual care strategy in improving outcomes. Trial Registration: clinicaltrials.gov Identifier: NCT01685840.

Effects of KCNQ2 gene truncation on M-type Kv7 potassium currents.

The KCNQ2 gene product, Kv7.2, is a subunit of the M-channel, a low-threshold voltage-gated K(+) channel that regulates mammalian and human neuronal excitability. Spontaneous mutations one of the KCNQ2 genes cause disorders of neural excitability such as Benign Familial Neonatal Seizures. However there appear to be no reports in which both human KCNQ2 genes are mutated. We therefore asked what happens to M-channel function when both KCNQ2 genes are disrupted. We addressed this using sympathetic neurons isolated from mice in which the KCNQ2 gene was truncated at a position corresponding to the second transmembrane domain of the Kv7.2 protein. Since homozygote KCNQ2-/- mice die postnatally, experiments were largely restricted to neurons from late embryos. Quantitative PCR revealed an absence of KCNQ2 mRNA in ganglia from KCNQ2-/- embryos but 100-120% increase of KCNQ3 and KCNQ5 mRNAs; KCNQ2+/- ganglia showed ∼30% less KCNQ2 mRNA than wild-type (+/+) ganglia but 40-50% more KCNQ3 and KCNQ5 mRNA. Neurons from KCNQ2-/- embryos showed a complete absence of M-current, even after applying the Kv7 channel enhancer, retigabine. Neurons from heterozygote KCNQ2+/- embryos had ∼60% reduced M-current. In contrast, M-currents in neurons from adult KCNQ2+/- mice were no smaller than those in neurons from wild-type mice. Measurements of tetraethylammonium block did not indicate an increased expression of Kv7.5-containing subunits, implying a compensatory increase in Kv7.2 expression from the remaining KCNQ2 gene. We conclude that mouse embryonic M-channels have an absolute requirement for Kv7.2 subunits for functionality, that the reduced M-channel activity in heterozygote KCNQ2+/- mouse embryos results primarily from a gene-dosage effect, and that there is a compensatory increase in Kv7.2 expression in adult mice.

Kv7/M-type potassium channels in rat skin keratinocytes.

Skin keratinocytes fulfil important signalling and protective functions. Immunocytochemical experiments revealed the unexpected presence of immunoreactivity for the M-type potassium channel subunit Kv7.2 in the keratinocyte layer of intact rat paw skin and in keratinocytes isolated from the skin of 1-day-old rats and cultured in vitro for 3-10 days. Application of the M-channel enhancer retigabine (3-10 µM) to isolated cultured rat keratinocytes: (a) increased outward membrane currents recorded under voltage clamp, (b) produced ~3 mV hyperpolarization at rest, (c) enhanced ~3-fold the release of ATP induced by the TRPV3 agonist carvacrol (1 mM) and (d) increased the amplitude of the carvacrol-induced intracellular Ca(2+) transient measured with Fura-2. The effect of retigabine on ATP release was prevented by the M-channel blocking agent XE991. We conclude that rat skin keratinocytes possess M-channels that, when activated, can modify their physiological properties, with potential significance for their sensory and other biological functions.

Functional significance of M-type potassium channels in nociceptive cutaneous sensory endings.

M-channels carry slowly activating potassium currents that regulate excitability in a variety of central and peripheral neurons. Functional M-channels and their Kv7 channel correlates are expressed throughout the somatosensory nervous system where they may play an important role in controlling sensory nerve activity. Here we show that Kv7.2 immunoreactivity is expressed in the peripheral terminals of nociceptive primary afferents. Electrophysiological recordings from single afferents in vitro showed that block of M-channels by 3 µM XE991 sensitized Aδ- but not C-fibers to noxious heat stimulation and induced spontaneous, ongoing activity at 32°C in many Aδ-fibers. These observations were extended in vivo: intraplantar injection of XE991 selectively enhanced the response of deep dorsal horn (DH) neurons to peripheral mid-range mechanical and higher range thermal stimuli, consistent with a selective effect on Aδ-fiber peripheral terminals. These results demonstrate an important physiological role of M-channels in controlling nociceptive Aδ-fiber responses and provide a rationale for the nocifensive behaviors that arise following intraplantar injection of the M-channel blocker XE991.

Distinct Nav1.7-dependent pain sensations require different sets of sensory and sympathetic neurons.

Human acute and inflammatory pain requires the expression of voltage-gated sodium channel Nav1.7 but its significance for neuropathic pain is unknown. Here we show that Nav1.7 expression in different sets of mouse sensory and sympathetic neurons underlies distinct types of pain sensation. Ablating Nav1.7 gene (SCN9A) expression in all sensory neurons using Advillin-Cre abolishes mechanical pain, inflammatory pain and reflex withdrawal responses to heat. In contrast, heat-evoked pain is retained when SCN9A is deleted only in Nav1.8-positive nociceptors. Surprisingly, responses to the hotplate test, as well as neuropathic pain, are unaffected when SCN9A is deleted in all sensory neurons. However, deleting SCN9A in both sensory and sympathetic neurons abolishes these pain sensations and recapitulates the pain-free phenotype seen in humans with SCN9A loss-of-function mutations. These observations demonstrate an important role for Nav1.7 in sympathetic neurons in neuropathic pain, and provide possible insights into the mechanisms that underlie gain-of-function Nav1.7-dependent pain conditions.

Some new insights into the molecular mechanisms of pain perception.

Bradykinin is the most potent endogenous inducer of acute pain. However, the way in which it excites nociceptive sensory nerve endings is still unclear. In an article recently published in the JCI, Liu et al. suggest a new mechanism via which bradykinin induces acute spontaneous pain. The authors report that the stimulation of B2 bradykinin receptors by bradykinin triggers the release of intracellular calcium ions from nociceptive sensory neurons of rat dorsal root ganglia. This depolarizes the sensory nerve endings by simultaneously closing M-type potassium channels and opening TMEM16A chloride channels, resulting in the production of nociceptive signals. Here, we discuss the relationship between this effect and a previously described mechanism for pain sensitization and evaluate its potential significance for therapeutic pain control. A separate study by Patwardhan et al. in this issue of the JCI identifies oxidized linoleic acid metabolites as novel mediators of thermally induced pain.

Neural KCNQ (Kv7) channels.

KCNQ genes encode five Kv7 K(+) channel subunits (Kv7.1-Kv7.5). Four of these (Kv7.2-Kv7.5) are expressed in the nervous system. Kv7.2 and Kv7.3 are the principal molecular components of the slow voltage-gated M-channel, which widely regulates neuronal excitability, although other subunits may contribute to M-like currents in some locations. M-channels are closed by receptors coupled to Gq such as M1 and M3 muscarinic receptors; this increases neuronal excitability and underlies some forms of cholinergic excitation. Muscarinic closure results from activation of phospholipase C and consequent hydrolysis and depletion of membrane phosphatidylinositol-4,5-bisphosphate, which is required for channel opening. Some effects of M-channel closure, determined from transmitter action, selective blocking drugs (linopirdine and XE991) and KCNQ2 gene disruption or manipulation, are as follows: (i) in sympathetic neurons: facilitation of repetitive discharges and conversion from phasic to tonic firing; (ii) in sensory nociceptive systems: facilitation of A-delta peripheral sensory fibre responses to noxious heat; and (iii) in hippocampal pyramidal neurons: facilitation of repetitive discharges, enhanced after-depolarization and burst-firing, and induction of spontaneous firing through a reduction of action potential threshold at the axon initial segment. Several drugs including flupirtine and retigabine enhance neural Kv7/M-channel activity, principally through a hyperpolarizing shift in their voltage gating. In consequence they reduce neural excitability and can inhibit nociceptive stimulation and transmission. Flupirtine is in use as a central analgesic; retigabine is under clinical trial as a broad-spectrum anticonvulsant and is an effective analgesic in animal models of chronic inflammatory and neuropathic pain.

Local translation in primary afferent fibers regulates nociception.

Recent studies have demonstrated the importance of local protein synthesis for neuronal plasticity. In particular, local mRNA translation through the mammalian target of rapamycin (mTOR) has been shown to play a key role in regulating dendrite excitability and modulating long-term synaptic plasticity associated with learning and memory. There is also increased evidence to suggest that intact adult mammalian axons have a functional requirement for local protein synthesis in vivo. Here we show that the translational machinery is present in some myelinated sensory fibers and that active mTOR-dependent pathways participate in maintaining the sensitivity of a subpopulation of fast-conducting nociceptors in vivo. Phosphorylated mTOR together with other downstream components of the translational machinery were localized to a subset of myelinated sensory fibers in rat cutaneous tissue. We then showed with electromyographic studies that the mTOR inhibitor rapamycin reduced the sensitivity of a population of myelinated nociceptors known to be important for the increased mechanical sensitivity that follows injury. Behavioural studies confirmed that local treatment with rapamycin significantly attenuated persistent pain that follows tissue injury, but not acute pain. Specifically, we found that rapamycin blunted the heightened response to mechanical stimulation that develops around a site of injury and reduced the long-term mechanical hypersensitivity that follows partial peripheral nerve damage--a widely used model of chronic pain. Our results show that the sensitivity of a subset of sensory fibers is maintained by ongoing mTOR-mediated local protein synthesis and uncover a novel target for the control of long-term pain states.

Dorsal root ganglion neurones in culture: a model system for identifying novel analgesic targets?

Ion channels represent attractive targets in the development of novel analgesics for the treatment of pain. Dorsal root ganglion (DRG) neurones in culture can share characteristics with nociceptors in vivo and are frequently used to investigate the ion channels that underlie the transduction of noxious stimuli into electrical activity during sensory processing. In this article, I describe the methods used to prepare cultures of DRG neurones including the procedures for the dissection, enzymatic dissociation and plating. The criteria used to identify putative nociceptors in vitro are reviewed and using the M-current as an example I highlight how potential analgesic targets can be identified by combining the use of the voltage clamp technique with the use of selective pharmacological agents.

KCNQ/M currents in sensory neurons: significance for pain therapy.

Neuronal hyperexcitability is a feature of epilepsy and both inflammatory and neuropathic pain. M currents [IK(M)] play a key role in regulating neuronal excitability, and mutations in neuronal KCNQ2/3 subunits, the molecular correlates of IK(M), have previously been linked to benign familial neonatal epilepsy. Here, we demonstrate that KCNQ/M channels are also present in nociceptive sensory systems. IK(M) was identified, on the basis of biophysical and pharmacological properties, in cultured neurons isolated from dorsal root ganglia (DRGs) from 17-d-old rats. Currents were inhibited by the M-channel blockers linopirdine (IC50, 2.1 microm) and XE991 (IC50, 0.26 microm) and enhanced by retigabine (10 microm). The expression of neuronal KCNQ subunits in DRG neurons was confirmed using reverse transcription-PCR and single-cell PCR analysis and by immunofluorescence. Retigabine, applied to the dorsal spinal cord, inhibited C and Adelta fiber-mediated responses of dorsal horn neurons evoked by natural or electrical afferent stimulation and the progressive "windup" discharge with repetitive stimulation in normal rats and in rats subjected to spinal nerve ligation. Retigabine also inhibited responses to intrapaw application of carrageenan in a rat model of chronic pain; this was reversed by XE991. It is suggested that IK(M) plays a key role in controlling the excitability of nociceptors and may represent a novel analgesic target.

Stoichiometry of expressed KCNQ2/KCNQ3 potassium channels and subunit composition of native ganglionic M channels deduced from block by tetraethylammonium.

KCNQ2 and KCNQ3 potassium-channel subunits can form both homomeric and heteromeric channels; the latter are thought to constitute native ganglionic M channels. We have tried to deduce the stoichiometric contributions of KCNQ2 and KCNQ3 subunits to currents generated by the coexpression of KCNQ2 and KCNQ3 cDNA plasmids in Chinese hamster ovary (CHO) cells, and to native M currents in dissociated rat superior cervical ganglion (SCG) neurons, by comparing the block of these currents produced by tetraethylammonium (TEA) with the block of currents generated by a tandem KCNQ3/2 construct. TEA concentration-inhibition curves against coexpressed KCNQ2 plus KCNQ3 currents, and against native M currents in SCG neurons from 6-week-old [postnatal day 45 (P45)] rats, were indistinguishable from those for the expressed tandem construct, and fully accorded with a 1:1 stoichiometry. Inhibition curves in neurons from younger (P17) rats could be better fitted assuming an additional small proportion of current carried by KCNQ2 homomultimers. Single-cell PCR yielded signals for KCNQ2, KCNQ3, and KCNQ5 mRNAs in all SCG neurons tested from both P17 and P45 rats. Quantitative PCR of whole-ganglion mRNA revealed stable levels of KCNQ2 and KCNQ5 mRNA between P7 and P45, but excess and incrementing levels of KCNQ3 mRNA. Increasing levels of KCNQ3 protein between P17 and P45 were confirmed by immunocytochemistry. We conclude that coexpressed KCNQ2 plus KCNQ3 cDNAs generate channels with 1:1 (KCNQ2:KCNQ3) stoichiometry in CHO cells and that native M channels in SCG neurons adopt the same conformation during development, assisted by the increased expression of KCNQ3 mRNA and protein.