In cardiac myocytes, multiple receptor mediated signalling pathways converge on cyclic nucleotide production. These second messengers act to achieve changes in cellular function. Despite this, each signalling molecule and receptor can achieve distinct sub-cellular effects. This has led to the theory of cyclic nucleotide compartmentation, which has been postulated to be mediated by phosphodiesterases (PDEs). Research in this field has focused on compartmentation using β-adrenergic stimulation. As an extension of this work, we investigated the effects of two agonists, prostaglandin E2 (PGE2; 10 nM) and forskolin (FSK; 30 nM), on various cellular parameters in the presence of either cilostamide (1 µM) a selective PDE3 inhibitor, or Ro 20-1724 (10 µM) a selective PDE4 inhibitor. In myocytes treated with PGE2, unloaded cell shortening and intracellular calcium transients exhibited significantly different (p<0.05) values of 147 ± 10% and 138 ± 5% of pre-treatment (t=0) values, respectively, in the presence of PGE2 and Ro 20-1724 (all n=5). However, values were not significantly different in cells pre-treated with cilostamide. Conversely, FSK resulted in significant increases of 153 ± 9% (n=5; P>0.05) and 189 ± 20% (n=5; P>0.05) of t=0 in cells treated with cilostamide and Ro 20-1724, respectively. PGE2 enhanced ICa,L was not altered using either PDE inhibitor. However, with FSK as an agonist, a significant increase in peak ICa,L from -6.0 ± 0.8 pA/pF to -7.7 ± 0.4 pA/pF (n=5; P>0.05) was observed in cells pre-treated with Ro 20-1724. SR calcium loading was also increased, but only in cells pre-treated with Ro 20-1724, with values of 127 ± 11% and 156 ± 47% of t=0 (n=5) for FSK and PGE2, respectively. Our results demonstrate that a unique pattern of regulation exists for PGE2 and that it is different from what was found previously with isoproterenol. We have shown that this is achieved by functionally localizing PDEs to distinct compartments. Specifically, PDE4 is localized at the SR, PDE3 at the sarcomere, and a combination of both at the calcium channel. However, our ICa,L results also indicate that the location of the receptor and adenylate cyclases must be considered relevant to compartmentalizing the cAMP signal. / Thesis (Master, Physiology) -- Queen's University, 2009-07-15 11:01:49.571

The electrical heterogeneity of the heart has been recognized as an important feature of normal cardiac function. In cardiac myocytes, considerable electrophysiological differences in sodium channel currents have been reported between the atria and the ventricle. Although, these differences have been primarily attributed to heterogeneous populations of Na+ channel isoforms within cardiac tissue, the link between these electrophysiological differences and certain cardiac pathologies has been loosely studied. We sought to further elucidate the electrophysiological differences between the atria and the ventricle by characterizing INa in both cell types. For these studies we had initially predicted the atria to contain a greater density of TTX-sensitive Na+ channel isoforms compared to that of the ventricle. We used two well-known Na+ channel blockers: lidocaine (100 μM, 30 μM, 10 μM) and tetrodotoxin (TTX; 10 nM, 30 nM). In addition, we also applied hydrogen peroxide (H2O2; 100 μM, 30 μM, 10 μM) to atrial myocytes, which served as our pathological model for reactive oxygen species (ROS). When we applied lidocaine to cardiac myocytes, we observed an overall mixed response in both cell types. Specifically, we noted the most significant differences (p < 0.05) in peak INa, shifts in steady-state inactivation, and impaired recovery from fast inactivation in the presence of 100 μM lidocaine. Given the non-uniform responses to lidocaine, our results support the theory that tissue specific populations of Na+ channel isoforms exist within cardiac myocytes. In order to further elucidate the electrophysiological differences between the ventricle and the atria, we applied TTX, which is selective for TTX-sensitive Na+ currents. Our results indicated no overall significant differences between the ventricle and the atria, suggesting that the population of TTX-sensitive Na+ channel isoforms within the atria specifically, may not be pharmacologically detectable. Finally, our results also demonstrated that the atria are sensitive to ROS, where H2O2 significantly prolonged the action potential duration (APD) in atrial myocytes. Our results also suggest that, in addition to INa, other ion channels may be mediating a component of the H2O2-induced prolongation of the APD in adult rat atrial myocytes. / Thesis (Master, Physiology) -- Queen's University, 2010-08-27 10:04:19.043

No description available.

Autophagy is a process by which cells re-cycle organelles and macromolecular proteins during cellular stress. Defects in the regulation of autophagy have been associated with various human pathologies including heart failure. In the heart tumor suppressor p53 protein is known to promote apoptotic and autophagic cell death. We found p53 over-expression increased endogenous protein level of the hypoxia-inducible Bcl-2 death gene Bnip3 which leads to loss of mitochondrial membrane potential (ΔΨm). This was accompanied by autophagic flux and cell death. Conversely, loss of function of Bnip3 in cardiac myocytes or Bnip3-/- mouse embryonic fibroblasts prevented mitochondrial targeting of p53 and autophagic cell death. These data provide the first evidence for the dual regulation of autophagic cell death of cardiac myocytes by p53 that is mutually dependent on Bnip3 activation. Hence, our findings may explain how autophagy and cell death are dually regulated during cardiac stress conditions where p53 is activated.

Autophagy is a process by which cells re-cycle organelles and macromolecular proteins during cellular stress. Defects in the regulation of autophagy have been associated with various human pathologies including heart failure. In the heart tumor suppressor p53 protein is known to promote apoptotic and autophagic cell death. We found p53 over-expression increased endogenous protein level of the hypoxia-inducible Bcl-2 death gene Bnip3 which leads to loss of mitochondrial membrane potential (ΔΨm). This was accompanied by autophagic flux and cell death. Conversely, loss of function of Bnip3 in cardiac myocytes or Bnip3-/- mouse embryonic fibroblasts prevented mitochondrial targeting of p53 and autophagic cell death. These data provide the first evidence for the dual regulation of autophagic cell death of cardiac myocytes by p53 that is mutually dependent on Bnip3 activation. Hence, our findings may explain how autophagy and cell death are dually regulated during cardiac stress conditions where p53 is activated.

Previous investigations have demonstrated that reactive oxygen species such as hydrogen peroxide (H2O2) have the ability to alter electrophysiological and mechanical properties of rat ventricular cardiac myocytes. However, despite the breadth of the literature, there is little definitive consensus on the cellular mechanisms. The purpose of this study, therefore, was to study the cellular mechanism of action of H2O2 and test whether H2O2-mediated affects were partially a result of reverse-mode Na+/Ca2+ exchanger (NCX) activity. Unloaded cell shortening, intracellular Ca2+ transients, caffeine-induced Ca2+ transients, L-type Ca2+ channel recordings, and action potential waveforms were recorded in the presence of combinations of different compounds including Cd2+, H2O2, and KB-R7943. H2O2 was found to cause significant positive inotropy by an increase in contractility of 80 ± 20 % (n=6) and an increased amplitude of Ca2+ transients by 24 ± 14 % (n=8), relative to pre-treatment values. Interestingly, H2O2 caused an increase in contractility even in the presence of Cd2+ block from 4 ± 1 % (n=9) to 15 ± 3 % (n=5) of resting cell length. Using caffeine pulse experiments to induce unloading of the sarcoplasmic reticulum (SR), we found that 100µM H2O2 did not significantly alter SR Ca2+ load. Under control conditions, H2O2 significantly increased L-type Ca2+ currents while this H2O2-induced increase was not observed in myocytes pretreated with Cd2+. Positive inotropy in the presence of H2O2 was blocked using 10µM KB-R7943, a selective reverse-mode inhibitor of the NCX. However, it was found that 10µM KB-R7943 alone altered action potential profile and suppressed normal contraction. Altogether, the major finding of this study is that H2O2 has the ability to enhance myocardial contractility, even under conditions of L-type Ca2+ channel inhibition, through a mechanism that likely involves reverse-mode of the NCX. / Thesis (Master, Physiology) -- Queen's University, 2009-03-31 14:00:34.21

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 3 uppsatser.

No description available.

No description available.

No / 1 Pharmacological inhibitors of protein kinase A (PKA) and protein phosphatases 1/2A were used to determine whether basal L-type Ca2+ current (ICa) observed in the absence of exogenous ß-adrenergic receptor stimulation is sustained by PKA-mediated phosphorylation. Amphotericin B was used to record whole-cell ICa in the perforated patch-clamp configuration. 2 Calyculin A and isoprenaline (both 1 ¿mol l¿1) increased basal ICa (P<0.05), whereas H-89 inhibited ICa in a concentration-dependent manner with an IC50 ~5 ¿mol l¿1. H-89 also inhibited the response to 1.0 ¿mol l¿1 isoprenaline, although relatively high concentrations (30 ¿mol l¿1) were required to achieve complete suppression of the response. 3 Double-pulse protocols were used to study the effects of 10 ¿mol l¿1 H-89 on time-dependent recovery of ICa from voltage-dependent inactivation as well as the steady-state gating of ICa. T0.5 (time for ICa to recover to 50% of the preinactivation amplitude) increased in the presence of H-89 (P<0.05) but was unaffected by calyculin A or isoprenaline. 4 Steady-state activation/inactivation properties of ICa were unaffected by 10 ¿mol l¿1 H-89 or 1 ¿mol l¿1 calyculin A, whereas isoprenaline caused a leftward shift in both curves so that V0.5 for activation and inactivation became more negative. 5 Data show that basal ICa is regulated by cAMP-PKA-mediated phosphorylation in the absence of externally applied ß-receptor agonists and that relatively high concentrations of H-89 are required to fully suppress the response to ß-adrenergic receptor stimulation, thereby limiting the value of H-89 as a useful tool in dissecting signalling pathways in intact myocytes.