Background: In heart failure, the release of calcium becomes erratic leading to the generation of arrhythmias. Dysregulated Zn2+ homeostasis occurs in chronic heart failure.Results: Zn2+ can directly activate RyR2, removing the dependence of Ca2+ for channel activation.Conclusion: Zn2+ shapes Ca2+ dynamics by directly interacting with and modulating RyR2 function.Significance: This highlights a new role for Zn2+ in cardiac excitation-contraction coupling.

Prolonged hyperglycaemia impairs vascular reactivity and inhibits voltage-activated K(+) (Kv) channels. We examined acute effects of altering glucose concentration on the activity and inhibition by endothelin-1 (ET-1) of Kv currents of freshly isolated rat arterial myocytes. Peak Kv currents recorded in glucose-free solution were reversibly reduced within 200 s by increasing extracellular glucose to 4 mm. This inhibitory effect of glucose was abolished by protein kinase C inhibitor peptide (PKC-IP), and Kv currents were further reduced in 10 mm glucose. In current-clamped cells, membrane potentials were more negative in 4 than in 10 mm glucose. In 4 mm d-glucose, 10 nm ET-1 decreased peak Kv current amplitude at +60 mV from 23.5 +/- 3.3 to 12.1 +/- 3.1 pA pF(-1) (n = 6, P < 0.001) and increased the rate of inactivation, decreasing the time constant around fourfold. Inhibition by ET-1 was prevented by PKC-IP. When d-glucose was increased to 10 mm, ET-1 no longer inhibited Kv current (n = 6). Glucose metabolism was required for prevention of ET-1 inhibition of Kv currents, since fructose mimicked the effects of d-glucose, while l-glucose, sucrose or mannitol were without effect. Endothelin receptors were still functional in 10 mm d-glucose, since pinacidil-activated ATP-dependent K(+) (K(ATP)) currents were reduced by 10 nm ET-1. This inhibition was nearly abolished by PKC-IP, indicating that endothelin receptors could still activate PKC in 10 mm d-glucose. These results indicate that changes in extracellular glucose concentration within the physiological range can reduce Kv current amplitude and can have major effects on Kv channel modulation by vasoconstrictors.

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ET-1 inhibits Kv channels of mesenteric ASM through activation of PKCalpha, while AngII does so through PKCepsilon. This implies that ET-1 and AngII target Kv channels of ASM through different pathways of PKC-interacting proteins, so each vasoconstrictor enables its distinct PKC isoenzyme to interact functionally with the Kv channel.

While it is well established that mortality risk after myocardial infarction (MI) increases in proportion to blood glucose concentration at the time of admission, it is unclear whether there is a direct, causal relationship. We investigated potential mechanisms by which increased blood glucose may exert cardiotoxicity. Using a Wistar rat or guinea-pig isolated cardiomyocyte model, we investigated the effects on cardiomyocyte function and electrical stability of alterations in extracellular glucose concentration. Contractile function studies using electric field stimulation (EFS), patch-clamp recording, and Ca2+ imaging were used to determine the effects of increased extracellular glucose concentration on cardiomyocyte function. Increasing glucose from 5 to 20 mM caused prolongation of the action potential and increased both basal Ca2+ and variability of the Ca2+ transient amplitude. Elevated extracellular glucose concentration also attenuated the protection afforded by ischemic preconditioning (IPC), as assessed using a simulated ischemia and reperfusion model. Inhibition of PKCα and β, using Gö6976 or specific inhibitor peptides, attenuated the detrimental effects of glucose and restored the cardioprotected phenotype to IPC cells. Increased glucose concentration did not attenuate the cardioprotective role of PKCε, but rather activation of PKCα and β masked its beneficial effect. Elevated extracellular glucose concentration exerts acute cardiotoxicity mediated via PKCα and β. Inhibition of these PKC isoenzymes abolishes the cardiotoxic effects and restores IPC-mediated cardioprotection. These data support a direct link between hyperglycemia and adverse outcome after MI. Cardiac-specific PKCα and β inhibition may be of clinical benefit in this setting.

ATP-sensitive potassium (K ATP ) channels are abundantly expressed in the myocardium.Although a definitive role for the channel remains elusive they have been implicated in the phenomenon of cardioprotection, but the precise mechanism is unclear. We set out to test the hypothesis that the channel protects by opening early during ischemia to shorten action potential duration and reduce electrical excitability thus sparing intracellular ATP. This could reduce reperfusion injury by improving calcium homeostasis.Using a combination of contractile function analysis, calcium fluorescence imaging and patch clamp electrophysiology in cardiomyocytes isolated from adult male Wistar rats, we demonstrated that the opening of sarcolemmal K ATP channels was markedly delayed after cardioprotective treatments; ischemic preconditioning, adenosine and PMA. This was due to the preservation of intracellular ATP for longer during simulated ischemia therefore maintaining sarcolemmal K ATP channels in the closed state for longer. As the simulated ischemia progressed, K ATP channels opened to cause contractile, calcium transient and action potential failure, however there was no indication of any channel activity early during simulated ischemia to impart an energy sparing hyperpolarization or action potential shortening.We present compelling evidence to demonstrate that early opening of sarcolemmal K ATP channels during simulated ischemia is not part of the protective mechanism imparted by ischemic preconditioning or other PKC-dependent cardioprotective stimuli. On the contrary, channel opening was actually delayed. We conclude that sarcolemmal K ATP channel opening is a consequence of ATP depletion, not a primary mechanism of ATP preservation in these cells. 3Highlights:• Opening of the SarcoK ATP channel was proposed to be cardioprotective• Channel opening was delayed after cardioprotective stimuli• Ca 2+ & ATP levels were maintained during ischemia independent of SarcoK ATP opening• Mitochondrial function preserved during ischemia, independent of SarcoK ATP opening• Early opening of SarcoK ATP is not involved in PKC-dependent cardioprotection

Functional KATP (ATP-sensitive potassium) channels are hetero-octamers of four Kir6 (inwardly rectifying potassium) channel subunits and four SUR (sulphonylurea receptor) subunits. Possible interactions between the C-terminal domain of SUR2A and Kir6.2 were investigated by co-immunoprecipitation of rat SUR2A C-terminal fragments with full-length Kir6.2 and by analysis of cloned KATP channel function and distribution in HEK-293 cells (human embryonic kidney 293 cells) in the presence of competing rSUR2A fragments. Three maltose-binding protein-SUR2A fusions, rSUR2A-CTA (rSUR2A residues 1254-1545), rSUR2A-CTB (residues 1254-1403) and rSUR2A-CTC (residues 1294-1403), were co-immunoprecipitated with full-length Kir6.2 using a polyclonal anti-Kir6.2 antiserum. A fourth C-terminal domain fragment, rSUR2A-CTD (residues 1358-1545) did not co-immunoprecipitate with Kir6.2 under the same conditions, indicating a direct interaction between Kir6.2 and a 65-amino-acid section of the cytoplasmic C-terminal region of rSUR2A between residues 1294 and 1358. ATP- and glibenclamide-sensitive K+ currents were decreased in HEK-293 cells expressing full-length Kir6 and SUR2 subunits that were transiently transfected with fragments rSUR2A-CTA, rSUR2A-CTC and rSUR2A-CTE (residues 1294-1359) compared with fragment rSUR2A-CTD or mock-transfected cells, suggesting either channel inhibition or a reduction in the number of functional KATP channels at the cell surface. Anti-KATP channel subunit-associated fluorescence in the cell membrane was substantially lower and intracellular fluorescence increased in rSUR2A-CTE expressing cells; thus, SUR2A fragments containing residues 1294-1358 reduce current by decreasing the number of channel subunits in the cell membrane. These results identify a site in the C-terminal domain of rSUR2A, between residues 1294 and 1358, whose direct interaction with full-length Kir6.2 is crucial for the assembly of functional KATP channels.

AimsMembrane potential is a key determinant of vascular tone and many vasodilators act through the modulation of ion channel currents [e.g. the ATP-sensitive potassium channel (KATP)] involved in setting the membrane potential. Adenylyl cyclase (AC) isoenzymes are potentially important intermediaries in such vasodilator signalling pathways. Vascular smooth muscle cells (VSMCs) express multiple AC isoenzymes, but the reason for such redundancy is unknown. We investigated which of these isoenzymes are involved in vasodilator signalling and regulation of vascular ion channels important in modulating membrane potential.Methods and resultsAC isoenzymes were selectively depleted (by >75%) by transfection of cultured VSMCs with selective short interfering RNA sequences. AC6 was the predominant isoenzyme involved in vasodilator-mediated cAMP accumulation in VSMCs, accounting for ∼60% of the total response to β-adrenoceptor (β-AR) stimulation. AC3 played a minor role in β-AR signalling, whereas AC5 made no significant contribution. AC6 was also the principal isoenzyme involved in β-AR-mediated protein kinase A (PKA) signalling (determined using the fluorescent biosensor for PKA activity, AKAR3) and the substantial β-AR/PKA-dependent enhancement of KATP current. KATP current was shown to play a vital role in setting the resting membrane potential and in mediating the hyperpolarization observed upon β-AR stimulation.ConclusionAC6, but not the closely related AC5, plays a principal role in vasodilator signalling and regulation of the membrane potential in VSMCs. These findings identify AC6 as a vital component in the vasodilatory apparatus central to the control of blood pressure.