Previous studies have suggested that phosphodiesterase (PDE) hydrolysis of cyclic adenosine monophosphate (cAMP) is important in the generation of specific and segregated cAMP signals within cells. The purpose of this study was to determine if PDE compartmentation was important in cardiac ventricular myocytes. Therefore, we investigated the effects of β-adrenergic (β-AD) stimulation with isoproterenol in the presence of cilostamide, a PDE3 inhibitor, or Ro 20-1724, a PDE4 inhibitor, on unloaded cell shortening, L-type calcium currents and intracellular calcium levels in freshly dissociated adult rat ventricular myocytes. PDE3 inhibition resulted in a 216 ± 17 % (n=8) increase in unloaded cell shortening after ten minutes of isoproterenol exposure, whereas isoproterenol produced a statistically smaller increase of 155 ± 12 % (n=8) in the presence of PDE4 inhibition. There was a non-significant trend for PDE4 inhibition to produce larger increases in calcium currents (179 ± 17 % (n=4) of controls) than PDE3 inhibition (155 ± 10 % (n=6) of controls). Both PDE3 and PDE4 inhibitors had similar effects on isoproterenol-stimulated increases of calcium transient amplitude with values of 209 ± 14 % (n=8) and 185 ± 12 % (n=8), respectively. Determination of sarcoplasmic reticulum (SR) calcium load using caffeine pulse experiments demonstrated that PDE4 inhibition and isoproterenol superfusion produced a statistically larger increase in SR-calcium loading (139 ± 9 % (n=6)) than PDE3 inhibition and isoproterenol superfusion (113 ± 9 % (n=6)). These results suggest that PDE3 may be active in proximity to the contractile apparatus of cardiac myocytes, whereas PDE4 may be localized in a domain consisting of the L-type calcium channel and junctional SR. Consequently, our study provides functional evidence for differential localization of PDE isoforms in cardiac myocytes. / Thesis (Master, Physiology) -- Queen's University, 2008-01-18 10:14:29.671 / CIHR OGS OGSST

No / Rapid exposure of cardiac muscle to high concentrations of caffeine releases Ca 2+ from the sarcoplasmic reticulum (SR). This Ca 2+ is then extruded from the cell by the Na +/Ca 2+ exchanger. Measurement of the current carried by the exchanger ( I Na/Ca) can therefore be used to estimate of the Ca 2+ content of the SR. Previous studies have shown that caffeine, however, can also inhibit K + currents. We therefore investigated whether the inhibitory effects of caffeine on these currents could contaminate measurements of I Na/Ca. Caffeine caused partial inhibition of the inward rectifier K + current ( I K1): the outward current at ¿40 mV was 1.15±0.24 pA/pF in control and decreased to 0.34±0.15 pA/pF in the presence of 10 mmol/l caffeine ( P<0.05, n=15). This was similar to the effect of caffeine on the holding current observed at ¿40 mV in the absence of K + channel block and could therefore account for the contaminating effects of caffeine observed during measurements of I Na/Ca. Moreover, caffeine also partially inhibited the transient outward ( I to) and the delayed rectifier ( I K) K + currents.

To this day heart failure with preserved ejection fraction (HFpEF) remains a poorly understood malady. Half of all heart failure (HF) cases are HFpEF, and the prevalence of HF is on the rise. Unlike HF with reduced ejection fraction, HFpEF has no treatment options and is often times difficult to diagnose because victims of HFpEF often have pre-existing conditions. Vascular endothelial growth factor (VEGF) has been implicated in maintaining myocardial health and is thought to play a role in HFpEF. We sought to test the hypothesis that VEGF-A plays a role in HFpEF in a hypertensive murine model of HFpEF. Using Western blot analysis we found that there was an up regulation of VEGF-A in the homogenized left ventricle (LV) of our HFpEF mice. Unexpectedly, there was a down regulation of VEGF-A in the homogenized tissue from the aorta in those mice. To study the circulating levels of VEGF in our HFpEF mice we used an ELISA. We found that our HFpEF mice had similar levels of circulating VEGF as our control. This suggests that VEGF has paracrine/autocrine role in our HFpEF model rather than endocrine, like our human data suggested. To identify the cells responsible for the expression profile we saw in the homogenized tissue data we looked at the response of adult rat ventricular myocytes (ARVM) and vascular smooth muscle cells (VSMC) to aldosterone stimulation at short (1hr) and long (24hr) time points at both physiological (50nm) and pathological (1μm) concentrations. To do this analysis we recruited the help of Western blot, ELISA and RT-PCR techniques to construct a consistent VEGF expression profile. The Western blot ARVM data showed statistically significant (P<0.05) increase in VEGF-A to pathological doses of aldosterone, especially at the longer time point. When we tested the VSMC using Western blot analysis, we found that the trend of our n=1 sample suggested a strong response to the physiological dose of aldosterone in the short term. Using the more sensitive ELISA technique to measure the VEGF content of our VCMS we increasing our sample size to n=4 and found no statistically significant (p=NS) response to aldosterone stimulation from the VSMC. However, looking at the trends in the data it is clear that VSMC increases VEGF in response to long-term physiological doses of aldosterone. This is contrary to what we found using Western blot analysis, so we queried the VEGF mRNA from the VSMC to settle the score. Unfortunately, this too proved fruitless. The RT-PCR data was not significant and the trend was that of the ARVM expression profile. We initially turned to VSMC because we hypothesized that they could contribute to the paracrine/autocrine activity similar to what we saw in the LV from the ARVM. It is unclear if VSMC play a role in HFpEF progression, but their lack of consistent response to aldosterone could potential explain the down regulation of VEGF-A we observed in the aorta of our HFpEF mice. We initially sough to test the hypothesis that VEGF-A plays a role in our HFpEF mouse model, what we found was that ARVM contribute to localized VEGF-A increased production in the LV while in the aorta there is a down regulation of VEGF-A in our HFpEF model, we are unable to make any conclusion about VSMC response to aldosterone because of insufficient sample size. Thus in conclusion, it appears that VEGF-A does play a role in our HFpEF model specifically in a paracrine/autocrine manner in the LV where the ARVM contributes to the increased production of the cytokine.

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a ubiquitously expressed membrane protein that regulates intracellular pH. In the myocardium, NHE1 has been implicated in ischemia/reperfusion (I/R) and cardiac hypertrophy (CH). Hormonal, autocrine and paracrine stimuli, acidosis, cardiotoxic metabolites released during I/R and CH increases NHE1 protein expression and activity. The involvement of NHE1 in CH and I/R has been further supported with the use of NHE1 inhibitors, which have been beneficial in the prevention/regression of several models of CH and I/R injury. Despite the fact that elevation of NHE1 expression and activity have been demonstrated in several models of heart disease, it was unclear whether elevation of NHE1 protein expression was sufficient to induce a specific cardiac pathology, or whether activation of the protein was required. To understand the direct role of NHE1 in CH and I/R, an in vivo and in vitro gain-of-function model, expressing varying levels and activities of NHE1 were examined. In vivo, our N-line mice expressed wild type NHE1 and our K-line mice expressed constitutively active NHE1. In vitro, neonatal rat ventricular cardiomyocytes were infected with the IRM adenovirus containing wild type NHE1 or the K-IRM adenovirus containing active NHE1. We demonstrated that expression of constitutively active NHE1 promotes CH to a much greater degree than expression of wild type NHE1 alone, both in vivo and in vitro. This NHE1-dependent hypertrophic response occurred independent of signaling pathways involved in CH including, mitogen activated protein kinases, p90 ribosomal S6 kinase, calcineurin and glycogen synthase kinase. The NHE1-dependent hypertrophic effect also occurred independent of gender. In addition, the expression of active NHE1 increased the susceptibility of intact mice to neurohormonal stimulation and progressed the hypertrophic response. When these hearts expressing active NHE1 were subjected to I/R using the ex vivo working heart perfusion model, fatty acid (FA) oxidation and glycolysis rates increased, thus generating greater ATP production rates. This was associated with cardioprotective effects in the myocardium, as well as a more energetically efficient myocardium. Expression of the endoplasmic reticulum (ER) stress response proteins, calreticulin and PDI were also shown to be increased relative to controls, and may contribute to the cardioprotection observed. We demonstrate that active NHE1 induces cardioprotection and alters cardiac metabolism in working hearts subjected to I/R. Overall, our results suggest that expression of active NHE1 has a double edged sword effect, on one side it induces CH while on the other side, it protects the heart against I/R injury.

We develop a composite multiphysics model of excitation-contraction coupling for a rat ventricular myocyte under voltage clamp (VC) conditions to: (1) probe mechanisms underlying the response to Ca2+-perturbation; (2) investigate the factors influencing its electromechanical response; and (3) examine its rate-dependent behavior (particularly the force-frequency response (FFR)). Motivation for the study was to pinpoint key control variables influencing calcium-induced calcium-release (CICR) and examine its role in the context of a physiological control system regulating cytosolic Ca2+ concentration and hence the cardiac contractile response. Our cell model consists of an electrical-equivalent model for the cell membrane and a fluid-compartment model describing the flux of ionic species between the extracellular and several intracellular compartments. The model incorporates frequency-dependent calmodulin (CaM) mediated spatially heterogenous interaction of calcineurin (CaN) and Ca2+/calmodulin-dependent protein kinase-II (CaMKII) with their principal targets and accounts for rate-dependent, cyclic adenosine monophosphate (cAMP)-mediated up-regulation. We also incorporate a biophysical model for cardiac contractile mechanics to study the factors influencing force response. The model reproduces measured VC data published by several laboratories, and generates graded Ca2+-release with high Ca2+ gain by achieving negative feedback control and Ca2+-homeostasis. We examine the dependence of cellular contractile response on: (1) the amount of activator Ca2+ available; (2) the type of mechanical load applied; (3) temperature (22 to 38ºC); and (4) myofilament Ca2+ sensitivity. We demonstrate contraction-relaxation coupling over a wide range of physiological perturbations. Our model reproduces positive peak FFR observed in rat ventricular myocytes and provides quantitative insight into the underlying rate-dependence of CICR. The role of Ca2+ regulating mechanisms are examined in handling induced Ca2+-perturbations using a rigorous cellular Ca2+ balance. Extensive testing of the composite model elucidates the importance of various direct and indirect modulatory influences on the cellular twitch-response with wide agreement with measured data on all accounts. We identify cAMP-mediated stimulation, and rate-dependent CaMKII-mediated up-regulation of Ca2+-trigger current (ICaL) as the key mechanisms underlying the aforementioned positive FFR. Our model provides biophysically-based explanations of phenomena associated with CICR and provides mechanistic insights into whole-cell responses to a wide variety of testing approaches used in studies of cardiac myofilament contractility.

No description available.

Cardiac accessory β-subunits are part of macromolecular ion channel complexes. They can modulate electrophysiological properties of resulting ion currents and action potentials and are supposed to contribute to cardiac disease e.g. arrhythmias or Brugada syndrome. In my thesis, we characterized the functions of dipeptidyl peptidase-like protein 10 (DPP10), a transmembrane β-subunit of cardiac Na+ and K+ channels. Previous studies revealed that DPP10 is expressed in human heart and acts as regulator of Kv channel kinetics. In electrophysiological experiments, we found that DPP10 modulates Ito through Kv4.3 channel complexes by accelerating current densities and the time course of activation, inactivation and recovery from inactivation. Interestingly, co-expression of DPP10 with Kv4.3 and KChIP2 in CHO cells induced a slowly inactivating fraction of Ito, providing evidence for a contribution of Ito on the sustained outward K+ current in cardiomyoctes. Until then, the sustained fraction of K+ currents was thought to be due to IKur. We further studied the contribution of Kv4-mediated Ito to total K+ currents in human atrial myocytes using 4-Aminopyridine to block IKur in combination with Heteropoda toxin 2 to block Kv4 channels. Using this approach, it was possible to separate an Ito fraction of about 19% contributing to the late current component. These data suggest that the generation of a sustained current component of Ito induced by DPP10 may affect the late repolarization phase of an atrial action potential. To further explore the functions of DPP10, we investigated a potential interaction with Nav channels in cardiomyocytes. It was possible to detect DPP10 in human ventricles, with higher expression levels in patients with heart failure. We demonstrated that DPP10 affects cellular action potentials in isolated rat cardiomyocytes after adenoviral gene transfer indicating a reduction in Na+ current density. Voltage-dependent Na+ channel activation and inactivation curve was shifted to more positive potentials with overexpression of DPP10, resulting in enhanced availability of Na+ channels for activation, along with increasing window Na+ current. Thus, we assumed a role of DPP10 on promotion of arrhythmias via interaction with Nav1.5. The results of this study can help to understand the complex interaction pattern between Nav and Kv channels and the role of their β-subunits, especially DPP10. In conclusion, DPP10 was identified as a new modulator of Kv and Nav currents in the human heart, suggesting that this β-subunit may contributes to cardiac arrhythmias and might be a new therapeutic target.:1 Introduction 1.1 The cardiac action potential 1.2 Cardiac potassium channels 1.2.1 The Kv4.3 channel complex 1.2.2 Accessory β subunits of K+ channel 1.2.3 The Kv1.5 channel 1.2.4 Separation of Ito and IKur in native cardiomyocytes 1.3 Cardiac sodium channels 1.3.1 Molecular construction of Nav1.5 channel 1.3.2 Accessory β subunits of Na+ channel 1.3.3 The role of Nav1.5 in cardiac electrical disorders 1.4 Aim of the thesis and systematic approach 2 The research articles 3 Summary 4 Zusammenfassung 6 References 7 Appendices 7.1 Abbreviations