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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

E2F-1 is a transcription factor that is involved in cellular growth and regulates the transition between G1 and S phase during the cell cycle. However, the mechanisms by which E2F-1 regulates endoplasmic reticulum (ER) stress in ventricular myocytes remain poorly defined. ER stress was triggered by tunicamycin or thapsigargin; gene transcription was assessed by polymerase chain reaction and protein expression was detected by western blot. Cell viability and mitochondrial defects were assessed by fluorescent microscopy imaging. During ER stress, E2F-1 repressed signaling molecules of the unfolded protein response (UPR) and sensitized myocytes to cell death triggered by thapsigargin that was inhibited in Bnip3 null fibroblasts. Bnip3Δex3 rescued thapsigargin-induced cardiac apoptosis, blocked mitochondrial defects and rescued hypoxia/ER stress induced cardiac cell death. This study provides evidence that E2F-1 sensitizes ventricular myocytes to ER stress induced apoptosis 1) by repressing the UPR; 2) that is Bnip3 dependent; and 3) mediated by mitochondrial dysfunction.

No description available.

E2F-1 is a transcription factor that is involved in cellular growth and regulates the transition between G1 and S phase during the cell cycle. However, the mechanisms by which E2F-1 regulates endoplasmic reticulum (ER) stress in ventricular myocytes remain poorly defined. ER stress was triggered by tunicamycin or thapsigargin; gene transcription was assessed by polymerase chain reaction and protein expression was detected by western blot. Cell viability and mitochondrial defects were assessed by fluorescent microscopy imaging. During ER stress, E2F-1 repressed signaling molecules of the unfolded protein response (UPR) and sensitized myocytes to cell death triggered by thapsigargin that was inhibited in Bnip3 null fibroblasts. Bnip3Δex3 rescued thapsigargin-induced cardiac apoptosis, blocked mitochondrial defects and rescued hypoxia/ER stress induced cardiac cell death. This study provides evidence that E2F-1 sensitizes ventricular myocytes to ER stress induced apoptosis 1) by repressing the UPR; 2) that is Bnip3 dependent; and 3) mediated by mitochondrial dysfunction.

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

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.

No description available.