BACKGROUND AND PURPOSEModulation of Kv7/M channel function represents a relatively new strategy to treat neuronal excitability disorders such as epilepsy and neuropathic pain. We designed and synthesized a novel series of pyrazolo [1,5-a] pyrimidin-7(4H)-one compounds, which activate Kv7 channels. Here, we characterized the effects of the lead compound, QO-58, on Kv7 channels and investigated its mechanism of action. EXPERIMENTAL APPROACHA perforated whole-cell patch technique was used to record Kv7 currents expressed in mammalian cell lines and M-type currents from rat dorsal root ganglion neurons. The effects of QO-58 in a rat model of neuropathic pain, chronic constriction injury (CCI) of the sciatic nerve, were also examined. KEY RESULTSQO-58 increased the current amplitudes, shifted the voltage-dependent activation curve in a more negative direction and slowed the deactivation of Kv7.2/Kv7.3 currents. QO-58 activated Kv7.1, Kv7.2, Kv7.4 and Kv7.3/Kv7.5 channels with a more selective effect on Kv7.2 and Kv7.4, but little effect on Kv7.3. The mechanism of QO-58's activation of Kv7 channels was clearly distinct from that used by retigabine. A chain of amino acids, Val , in Kv7.2 was important for QO-58 activation of this channel. QO-58 enhanced native neuronal M currents, resulting in depression of evoked action potentials. QO-58 also elevated the pain threshold of neuropathic pain in the sciatic nerve CCI model. CONCLUSIONS AND IMPLICATIONSThe results indicate that QO-58 is a potent modulator of Kv7 channels with a mechanism of action different from those of known Kv7 openers. Hence, QO-58 shows potential as a treatment for diseases associated with neuronal hyperexcitability. AbbreviationsBFNC, benign familial neonatal convulsions; CCI, chronic constriction injury; DRG, dorsal root ganglion; PPOs, pyrazolo[1,5-a] pyrimidin-7(4H)-ones; RTG, retigabine; SAR, structure-activity relationship

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Voltage-gated Kv1.1 potassium channel α-subunits, encoded by the Kcna1 gene, have traditionally been regarded as neural-specific with no expression or function in the heart. However, recent data revealed that Kv1.1 subunits are expressed in atria where they may have an overlooked role in controlling repolarization and arrhythmia susceptibility independent of the nervous system. To explore this concept in more detail and to identify functional and molecular effects of Kv1.1 channel impairment in the heart, atrial cardiomyocyte patch-clamp electrophysiology and gene expression analyses were performed using Kcna1 knockout ( Kcna1−/−) mice. Specifically, we hypothesized that Kv1.1 subunits contribute to outward repolarizing K+ currents in mouse atria and that their absence prolongs cardiac action potentials. In voltage-clamp experiments, dendrotoxin-K (DTX-K), a Kv1.1-specific inhibitor, significantly reduced peak outward K+ currents in wild-type (WT) atrial cells but not Kcna1−/− cells, demonstrating an important contribution by Kv1.1-containing channels to mouse atrial repolarizing currents. In current-clamp recordings, Kcna1−/− atrial myocytes exhibited significant action potential prolongation which was exacerbated in right atria, effects that were partially recapitulated in WT cells by application of DTX-K. Quantitative RT-PCR measurements showed mRNA expression remodeling in Kcna1−/− atria for several ion channel genes that contribute to the atrial action potential including the Kcna5, Kcnh2, and Kcnj2 potassium channel genes and the Scn5a sodium channel gene. This study demonstrates a previously undescribed heart-intrinsic role for Kv1.1 subunits in mediating atrial repolarization, thereby adding a new member to the already diverse collection of known K+ channels in the heart.

Oxidative stress drives the pathogenesis of atrial fibrillation (AF), the most common arrhythmia. In the cardiovascular system, cystathionine γ-lyase (CSE) serves as the primary enzyme producing hydrogen sulfide (H 2 S), a mammalian gasotransmitter that reduces oxidative stress. Using a case control study design in patients with and without AF and a mouse model of CSE knockout (CSE-KO), we evaluated the role of H 2 S in the etiology of AF. Patients with AF (n = 51) had significantly reduced plasma acid labile sulfide levels compared to patients without AF (n = 65). In addition, patients with persistent AF (n = 25) showed lower plasma free sulfide levels compared to patients with paroxysmal AF (n = 26). Consistent with an important role for H 2 S in AF, CSE-KO mice had decreased atrial sulfide levels, increased atrial superoxide levels, and enhanced propensity for induced persistent AF compared to wild type (WT) mice. Rescuing H 2 S signaling in CSE-KO mice by Diallyl trisulfide (DATS) supplementation or reconstitution with endothelial cell specific CSE over-expression significantly reduced atrial superoxide, increased sulfide levels, and lowered AF inducibility. Lastly, low H 2 S levels in CSE KO mice was associated with atrial electrical remodeling including longer effective refractory periods, slower conduction velocity, increased myocyte calcium sparks, and increased myocyte action potential duration that were reversed by DATS supplementation or endothelial CSE overexpression. Our findings demonstrate an important role of CSE and H 2 S bioavailability in regulating electrical remodeling and susceptibility to AF.

Background:The mechanism and significance of phosphoinositide metabolism during heart stress stimulations are not clear. Results: Norepinephrine and angiotensin II increase cardiac phosphatidylinositol 4,5-bisphosphate levels via an enhanced interaction between phosphatidylinositol 4-kinase III␤ and PKC, which correlate with a maintained systolic function. Conclusion: Cardiac phosphoinositide turnover is enhanced. Significance: A novel mechanism of phosphoinositide metabolism is described for modulation of cardiac function.

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Background/Aims: The slow component of the delayed rectifier K⁺ current (I_Ks) is one of the major repolarizing currents in the heart. Yet, the signaling mechanisms for norepinephrine- and angiotensin II-induced modulation of I_Ks in cardiac myocytes are far from being well understood. Methods: The whole-cell patch clamp technique was used to study the effects of norepinephrine and angiotensin II on I_Ks in guinea pig cardiac myocytes. Results: Both the α₁- and β-adrenoceptor inhibitors attenuated norepinephrine-induced enhancement of I_Ks, which was also significantly depressed by inhibitors of protein kinase A and C. Angiotensin II-induced inhibition of the I_Ks was inhibited by angiotensin type 1 receptor blocker losartan and protein kinase C inhibitor. Conclusions: Norepinephrine and angiotensin II modulated I_Ks with opposite effects and distinct mechanisms. The activation of protein kinase A was the major component of the norepinephrine-induced activation of I_Ks while the activation of protein kinase C was responsible for the angiotensin II-induced inhibition of I_Ks. There was crosstalk between the α₁- and β-adrenoceptor that also contributed to the norepinephrine-induced enhancement of I_Ks. This current study provides new insight into the cellular signaling mechanisms of norepinephrine and angiotensin II, the two important modulators of cardiovascular function.