Chronic myocardial infarction promotes atrial action potential alternans, afterdepolarisations and fibrillation

Aims: Atrial fibrillation (AF) is increased in patients with heart failure resulting from myocardial infarction (MI). We aimed to determine the effects of chronic ventricular MI in rabbits on the susceptibility to AF, and underlying atrial electrophysiological and Ca2+-handling mechanisms. Methods and results: In Langendorff-perfused rabbit hearts, under beta-adrenergic-stimulation with isoproterenol (1 µM; ISO), 8 weeks MI decreased AF threshold, indicating increased AF-susceptibility. This was associated with increased atrial action potential duration-alternans at 90% repolarisation, by 147%, and no significant change in mean APD or atrial global conduction velocity (n=6-13 non-MI hearts, 5-12 MI). In atrial isolated myocytes, also under beta-stimulation, L-type Ca2+ current (ICaL) density and intracellular Ca2+-transient amplitude were decreased by MI, by 35% and 41%, respectively, and the frequency of spontaneous depolarisations (SDs) was substantially increased. MI increased atrial myocyte size and capacity, and markedly decreased transverse-tubule density. In non-MI hearts perfused with ISO, the ICaL-blocker nifedipine, at a concentration (0.02 µM) causing an equivalent ICaL-reduction (35%) to that from the MI, did not affect AF-susceptibility, and decreased APD. Conclusion: chronic MI in rabbits remodels atrial structure, electrophysiology and intracellular Ca2+-handling. Increased susceptibility to AF by MI, under beta-adrenergic-stimulation, may result from associated production of atrial APD-alternans and SDs, since steady-state APD and global conduction velocity were unchanged under these conditions, and may be unrelated to the associated reduction in whole-cell ICaL. Future studies may clarify potential contributions of local conduction changes, and cellular and sub-cellular mechanisms of alternans, to the increased AF-susceptibility

PAX4 Enhances Beta-Cell Differentiation of Human Embryonic Stem Cells

Background Human embryonic stem cells (HESC) readily differentiate into an apparently haphazard array of cell types, corresponding to all three germ layers, when their culture conditions are altered, for example by growth in suspension as aggregates known as embryoid bodies (EBs). However, this diversity of differentiation means that the efficiency of producing any one particular cell type is inevitably low. Although pancreatic differentiation has been reported from HESC, practicable applications for the use of β-cells derived from HESC to treat diabetes will only be possible once techniques are developed to promote efficient differentiation along the pancreatic lineages. Methods and Findings Here, we have tested whether the transcription factor, Pax4 can be used to drive the differentiation of HESC to a β-cell fate in vitro. We constitutively over-expressed Pax4 in HESCs by stable transfection, and used Q-PCR analysis, immunocytochemistry, ELISA, Ca2+ microfluorimetry and cell imaging to assess the role of Pax4 in the differentiation and intracellular Ca2+ homeostasis of β-cells developing in embryoid bodies produced from such HESC. Cells expressing key β-cell markers were isolated by fluorescence-activated cell sorting after staining for high zinc content using the vital dye, Newport Green. Conclusion Constitutive expression of Pax4 in HESC substantially enhances their propensity to form putative β-cells. Our findings provide a novel foundation to study the mechanism of pancreatic β-cells differentiation during early human development and to help evaluate strategies for the generation of purified β-cells for future clinical applications

Balanced changes in Ca buffering by SERCA and troponin contribute to Ca handling during β-adrenergic stimulation in cardiac myocytes

AIMS: During activation of cardiac myocytes, less than 1% of cytosolic Ca is free; the rest is bound to buffers, largely SERCA, and troponin C. Signalling by phosphorylation, as occurs during β-adrenergic stimulation, changes the Ca-binding affinity of these proteins and may affect the systolic Ca transient. Our aim was to determine the effects of β-adrenergic stimulation on Ca buffering and to differentiate between the roles of SERCA and troponin. METHODS AND RESULTS: Ca buffering was studied in cardiac myocytes from mice: wild-type (WT), phospholamban-knockout (PLN-KO), and mice expressing slow skeletal troponin I (ssTnI) that is not protein kinase A phosphorylatable. WT cells showed no change in Ca buffering in response to the β-adrenoceptor agonist isoproterenol (ISO). However, ISO decreased Ca buffering in PLN-KO myocytes, presumably unmasking the role of troponin. This effect was confirmed in WT cells in which SERCA activity was blocked with the application of thapsigargin. In contrast, ISO increased Ca buffering in ssTnI cells, presumably revealing the effect of an increase in Ca binding to SERCA. CONCLUSIONS: These data indicate the individual roles played by SERCA and troponin in Ca buffering during β-adrenergic stimulation and that these two buffers effectively counterbalance each other so that Ca buffering remains constant during β-adrenergic stimulation, a factor which may be physiologically important. This study also emphasizes the importance of taking into account Ca buffering, particularly in disease states where Ca binding to myofilaments or SERCA may be altered

Temporal Development of Autonomic Dysfunction in Heart Failure:Effects of Age in an Ovine Rapid-pacing Model

Heart failure (HF) is predominantly a disease of older adults and characterized by extensive sympatho-vagal imbalance leading to impaired reflex control of heart rate (HR). However, whether aging influences the development or extent of the autonomic imbalance in HF remains unclear. To address this, we used an ovine model of aging with tachypacing-induced HF to determine whether aging affects the chronotropic and inotropic responses to autonomic stimulation and reduction in heart rate variability (HRV) in HF. We find that aging is associated with increased cardiac dimensions and reduced contractility before the onset of tachypacing, and these differences persist in HF. Additionally, the chronotropic response to β-adrenergic stimulation was markedly attenuated in HF, and this occurred more rapidly in aged animals. By measuring HR during sequential autonomic blockade, our data are consistent with a reduced parasympathetic control of resting HR in aging, with young HF animals having an attenuated sympathetic influence on HR. Time-domain analyses of HR show a reduction in HRV in both young and aged failing animals, although HRV is lowest in aged HF. In conclusion, aging is associated with altered autonomic control and β-adrenergic responsiveness of HR, and these are exacerbated with the development of HF