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

BACKGROUND AND PURPOSEWe investigated the hypothesis that elevated glucose increases contractile responses in vascular smooth muscle and that this enhanced constriction occurs due to the glucose-induced PKC-dependent inhibition of voltage-gated potassium channels. EXPERIMENTAL APPROACHPatch-clamp electrophysiology in rat isolated mesenteric arterial myocytes was performed to investigate the glucose-induced inhibition of voltage-gated potassium (K v ) current. To determine the effects of glucose in whole vessel, wire myography was performed in rat mesenteric, porcine coronary and human internal mammary arteries. KEY RESULTSGlucose-induced inhibition of K v was PKC-dependent and could be pharmacologically dissected using PKC isoenzyme-specific inhibitors to reveal a PKCβ-dependent component of K v inhibition dominating between 0 and 10 mM glucose with an additional PKCα-dependent component becoming evident at concentrations greater than 10 mM. These findings were supported using wire myography in all artery types used, where contractile responses to vessel depolarization and vasoconstrictors were enhanced by increasing bathing glucose concentration, again with evidence for distinct and complementary PKCα/PKCβ-mediated components. CONCLUSIONS AND IMPLICATIONSOur results provide compelling evidence that glucose-induced PKCα/PKCβ-mediated inhibition of K v current in vascular smooth muscle causes an enhanced constrictor response. Inhibition of K v current causes a significant depolarization of vascular myocytes leading to marked vasoconstriction. The PKC dependence of this enhanced constrictor response may present a potential therapeutic target for improving microvascular perfusion following percutaneous coronary intervention after myocardial infarction in hyperglycaemic patients. Significant fluctuations in plasma glucose concentration also occur physiologically through the diurnal cycle of feeding and fasting, and such changes can be exaggerated under certain pathophysiological circumstances (e.g. type 1 or type 2 diabetes). According to NICE guidelines, diabetes is often associated with cardiovascular complications, including coronary artery disease (leading to myocardial infarction and angina), peripheral artery disease (leg claudication and gangrene) and carotid artery disease (strokes and dementia). There are also microvascular complications caused by diabetes, such as diabetic retinopathy, kidney and nerve damage (NICE guidelines, 2014). Recent evidence suggests that the plasma concentration of blood glucose may also play a significant role in enhancing vasoconstriction and so have a deleterious effect on microvascular reperfusion following percutaneous coronary intervention (Iwakura et al., 2003). The risk associated with these complications can be minimized by tight glycaemic control, although there is a need for therapies to reduce the risk further. Acute hyperglycaemia (15 mM), in healthy human subjects, increases systolic and diastolic BP and heart rate and decreases leg blood flow and blood viscosity (Gi...