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Title: The effects of stimulation frequency on intracellular calcium handling within the sheep atrial myocyte
Author: Wrigley, Daniel
ISNI:       0000 0004 5369 9337
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2015
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Atrial fibrillation (AF), is characterised by rapid, irregular atrial stimulation, which leads to reduced contraction, resulting in a negative force-frequency-relationship (FFR). It is thought that rapid stimulation frequencies, which are synonymous with AF, disturb excitation-contraction-coupling (ECC). Ageing increases both the prevalence and economic impact of AF progressively. Although mechanisms underlying AF have been investigated in human and animal models, the cellular electrophysiology, and molecular changes that render the atria in aged individuals more susceptible to AF, still remain poorly understood. To our knowledge, there are no data investigating the effects of rapid stimulation frequencies on the Ca2+ handling within atrial myocytes, of a larger mammal, which are able to maintain AF, and is physiologically relevant to the human. To investigate this, sheep atrial myocytes were isolated, via enzymatic digestion. Measurements of intracellular Ca2+ ([Ca2+]i) were obtained via analysis of fluorescence (Fluo 5F am). Electrophysiological experiments were performed via the perforated-patch clamp technique, under voltage-clamp stimulation, to assess measurements of [Ca2+]i and trans-sarcolemmal currents. All voltage clamp experiments were performed at 37°C. Analysis of CaMKII inhibition, on Ca2+ wave frequency, was performed in non-stimulated atrial myocytes, at room temperature. Increased stimulation frequency (from 1Hz – 5Hz) had a significant impact on Ca2+ handling and trans-sarcolemmal currents within the atria. A reduction in Ca2+ transient amplitude was observed with increased rate. This was achieved despite increases in SR Ca2+ content, which were due to increased SERCA activity. The reduction in Ca2+ transient amplitude was attributed to reduced L-type Ca2+ current (ICa-L). Ageing augmented the rise in diastolic [Ca2+]i which was observed with rate, but with no further impac on Ca2+ transient amplitude. The rate dependent increase in SR Ca2+ content was augmented with age, and was presumed to be as a result of a reduction in ICa-L.By sensitising the ryanodine receptor (RyR), with low-dose caffeine (500 μmol.l-1), the fractional release of the first Ca2+ transient upon application was exacerbated by an increase in rate. This rapidly decayed to control levels at all stimulation frequencies. This data suggests that increases in RYR sensitivity lead to a greater Ca2+ release from the SR, for a given trigger. By ceasing stimulation there was potentiation of the first Ca2+ transient, post-rest, in comparison to pre-rest at 1Hz. This was augmented by increased rate. As ICa-L was unaltered between pre- and post-rest, within each frequency, it was assumed that the increased SR Ca2+ content with rate, coupled with continued SR Ca2+ uptake during the rest period, enhanced the fractional SR Ca2+ release for a given trigger, thus potentiating the amplitude of the Ca2+ transient. However, this requires further investigation. Other data found that CaMKII inhibition (via KN93) had no effect on Ca2+ wave frequency in control, or heart failure (HF), non-stimulated sheep atrial myocytes, which suggests either CaMKII is not up-regulated in the HF model used, or the concentration of KN93 used was insufficient. Further investigation is required in this area. The alterations in the mechanisms that modulate SR Ca2+ release and uptake are affected by alterations in stimulation frequency, which alter key modulators of contractile force.
Supervisor: Not available Sponsor: BBSRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Calcium ; Atria ; Frequency