The cGMP-Dependent Protein Kinase II Is an Inhibitory Modulator of the Hyperpolarization-Activated HCN2 Channel

Opening of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is facilitated by direct binding of cyclic nucleotides to a cyclic nucleotide-binding domain (CNBD) in the C-terminus. Here, we show for the first time that in the HCN2 channel cGMP can also exert an inhibitory effect on gating via cGMP-dependent protein kinase II (cGKII)-mediated phosphorylation. Using coimmunoprecipitation and immunohistochemistry we demonstrate that cGKII and HCN2 interact and colocalize with each other upon heterologous expression as well as in native mouse brain. We identify the proximal C-terminus of HCN2 as binding region of cGKII and show that cGKII phosphorylates HCN2 at a specific serine residue (S641) in the C-terminal end of the CNBD. The cGKII shifts the voltage-dependence of HCN2 activation to 2–5 mV more negative voltages and, hence, counteracts the stimulatory effect of cGMP on gating. The inhibitory cGMP effect can be either abolished by mutation of the phosphorylation site in HCN2 or by impairing the catalytic domain of cGKII. By contrast, the inhibitory effect is preserved in a HCN2 mutant carrying a CNBD deficient for cGMP binding. Our data suggest that bidirectional regulation of HCN2 gating by cGMP contributes to cellular fine-tuning of HCN channel activity

Determination of Baroreflex Sensitivity during the Modified Oxford Maneuver by Trigonometric Regressive Spectral Analysis

BACKGROUND: Differences in spontaneous and drug-induced baroreflex sensitivity (BRS) have been attributed to its different operating ranges. The current study attempted to compare BRS estimates during cardiovascular steady-state and pharmacologically stimulation using an innovative algorithm for dynamic determination of baroreflex gain. METHODOLOGY/PRINCIPAL FINDINGS: Forty-five volunteers underwent the modified Oxford maneuver in supine and 60° tilted position with blood pressure and heart rate being continuously recorded. Drug-induced BRS-estimates were calculated from data obtained by bolus injections of nitroprusside and phenylephrine. Spontaneous indices were derived from data obtained during rest (stationary) and under pharmacological stimulation (non-stationary) using the algorithm of trigonometric regressive spectral analysis (TRS). Spontaneous and drug-induced BRS values were significantly correlated and display directionally similar changes under different situations. Using the Bland-Altman method, systematic differences between spontaneous and drug-induced estimates were found and revealed that the discrepancy can be as large as the gain itself. Fixed bias was not evident with ordinary least products regression. The correlation and agreement between the estimates increased significantly when BRS was calculated by TRS in non-stationary mode during the drug injection period. TRS-BRS significantly increased during phenylephrine and decreased under nitroprusside. CONCLUSIONS/SIGNIFICANCE: The TRS analysis provides a reliable, non-invasive assessment of human BRS not only under static steady state conditions, but also during pharmacological perturbation of the cardiovascular system

Cardiopoietic cell therapy for advanced ischemic heart failure: results at 39 weeks of the prospective, randomized, double blind, sham-controlled CHART-1 clinical trial

Cardiopoietic cells, produced through cardiogenic conditioning of patients' mesenchymal stem cells, have shown preliminary efficacy. The Congestive Heart Failure Cardiopoietic Regenerative Therapy (CHART-1) trial aimed to validate cardiopoiesis-based biotherapy in a larger heart failure cohort

cGMP-inhibited phosphodiesterase of cAMP (PDE3) and protein kinase a (PKA) play an important role in eliciting the positive chronotropic effect of Nitric Oxide (NO) donors

Background: NO donors exert a positive chronotropic effect by stimulating the pacemaker current If in sino-atrial node cells via a mechanism requiring guanylyl cyclase (GC) and cGMP, and involving mobilization of sarcoplasmic (SR) Ca2+. Cyclic GMP can directly stimulate If but it is ∼10-fold less potent than the 'physiological' If-gating nucleotide, cAMP. In ventricular myocytes, NO can increase cAMP levels through a cGMP-dependent inhibition of PDE3. Aim: To elucidate the role of PDE3, PKA and PKG in mediating the NO-If-dependent increase in HR. Methods and Results: In isolated guinea-pig atria with basal HR (B/L) of 174±3 bpm (n=69), we evaluated the HR response to increasing concentrations (from 0.1 to 100μmol/L) of the NO donors DEA-NO (n=8) or SIN-1 (+SOD 100 U/mL, n=6) or to 10 μmol/L SNP (n=9), (1) alone, and (2) after pretreatment (ca. 40min) with inhibitors of (i) PDE3 (milrinone, trequinsin or Ro-13-6482, n=22) or (ii) PKA (KT5720 or H-89, n=15) or (iii) PKG (KT5823 or Rp-8-pCPT-cGMPs, n=16). Whereas PKG inhibition had no effect on the magnitude of the increase in HR with DEA-NO (see Fig; bars show the peak increase in HR, mean± SEM; =p<0.05 vs. B/L, †=p<0.05 vs. the HR response DEA-NO alone), inhibition of PDE3 or PKA markedly attenuated the HR response to DEA-NO (and, similarly, to SIN-1 or SNP; not shown). Conclusion: cGMP-PDE3-cAMP signalling plays a significant part in eliciting the 'direct' positive chronotropic response to NO donors. Since PKA is known to stimulate SR Ca2+ release by phosphorylating ryanodine receptors, the present findings provide an important link between the activation of GC with NO donors and the NO-dependent mobilization of SR Ca2+ in SA node which we reported previously. (Graph Presented)