Increased Ca buffering underpins remodelling of Ca2+ handling in old sheep atrial myocytes

Abstract

Key points: Ageing is associated with an increased risk of cardiovascular disease and arrhythmias, with the most common arrhythmia being found in the atria of the heart. Little is known about how the normal atria of the heart remodel with age and thus why dysfunction might occur. We report alterations to the atrial systolic Ca²⁺ transient that have implications for the function of the atrial in the elderly. We describe a novel mechanism by which increased Ca buffering can account for changes to systolic Ca²⁺ in the old atria. The present study helps us to understand how the processes regulating atrial contraction are remodelled during ageing and provides a basis for future work aiming to understand why dysfunction develops.

Abstract: Many cardiovascular diseases, including those affecting the atria, are associated with advancing age. Arrhythmias, including those in the atria, can arise as a result of electrical remodelling or alterations in Ca²⁺ homeostasis. In the atria, age-associated changes in the action potential have been documented. However, little is known about remodelling of intracellular Ca²⁺ homeostasis in the healthy aged atria. Using single atrial myocytes from young and old Welsh Mountain sheep, we show the free Ca²⁺ transient amplitude and rate of decay of systolic Ca²⁺ decrease with age, whereas sarcoplasmic reticulum (SR) Ca content increases. An increase in intracellular Ca buffering explains both the decrease in Ca²⁺ transient amplitude and decay kinetics in the absence of any change in sarcoendoplasmic reticulum calcium transport ATPase function. Ageing maintained the integrated Ca²⁺ influx via I_Ca-L but decreased peak I_Ca-L . Decreased peak I_Ca-L was found to be responsible for the age-associated increase in SR Ca content but not the decrease in Ca²⁺ transient amplitude. Instead, decreased peak I_Ca-L offsets increased SR load such that Ca²⁺ release from the SR was maintained during ageing. The results of the present study highlight a novel mechanism by which increased Ca buffering decreases systolic Ca²⁺ in old atria. Furthermore, for the first time, we have shown that SR Ca content is increased in old atrial myocytes.

Keywords: age; atria; buffering; calcium.

Figure 1. Ageing increases cell size and decreases Ca²⁺ transient amplitude and rate of decay of systolic Ca²⁺ in the atria

A, confocal images of typical atrial myocytes from young and old sheep loaded with calcein‐AM. B, mean cellular capacitance of atrial myocytes from young and old animals (n = 40–53 cells, 16–20 animals). C, representative systolic Ca²⁺ transients from young and old atrial myocytes elicited under voltage clamp control. D, mean systolic Ca²⁺ transient amplitude calculated from data similar to (C) (n = 29–51 cells, 15–23 animals). E, typical experimental time course for Ca²⁺ including application of 10 mm caffeine. F, example normalized systolic Ca²⁺ transients. G, mean rate of decay of the systolic Ca²⁺ transient (k _SYS) (n = 29–51 cells, 15–23 animals). H, normalized caffeine‐evoked Ca²⁺ transients. I, mean data summarizing SERCA (k _SR) and sarcolemmal (k _CAFF)‐dependent rates of Ca²⁺ removal (n = 22–38 cells, 11–20 animals). ^* P < 0.05.

Figure 2. Expression levels and phosphorylation status of key SR related proteins are unaltered with age in the atria

A–D, representative western blots for SERCA2a, total PLB and phosphorylated PLB (at Ser16 and Thr17 sites). E and F, representative western blots for PP1 and PP2A and G and H, RyR2 including phosphorylation at both Ser2808 and Ser2814 and calsequestrin (CSQ; I). Mean data are shown relative to the internal control for three repeats of 7–10 animals per group. NS, not significant.

Figure 3. Both Ca buffering and SR Ca content are increased in old atrial myocytes

A, quantitative assessment of free and total Ca in young (black) and old (grey) atrial myocytes. Caffeine‐evoked free Ca²⁺ transients (top) are associated with inward I _NCX (middle), which was integrated in a cumulative manner and corrected for cell volume and Ca²⁺ removal via pathways other than NCX to give a measure of total Ca (lower). B, typical cellular buffer curves showing the relationship between free and total Ca fit with a hyperbolic function (27–40 cells, 18–21 animals). C, typical relationships between the differential of the falling phase of total Ca plotted with respect to free Ca²⁺. Data are shown for both young and old systolic and caffeine‐evoked Ca²⁺ transients. Data points were fit with linear regressions. D, mean SERCA‐dependent slope calculated by subtracting the slope of the relationship for the caffeine‐evoked Ca²⁺ transient from that of the systolic transient as shown in (C) (n = 5–11 cells, 4–8 animals). E, mean SR Ca content as calculated from (A) (n = 43–52 cells, 23–24 animals). F – H describe the calculation of correction factors necessary to accurately calculate SR Ca content. F, cellular volume, calculated by confocal sections through a calcein loaded cell, plotted against the measured cellular capacitance for young (black triangles) and old (grey cross) cells. Young and old data sets were fit by linear regressions that were not significantly different for age. The dashed line therefore represents the fit to the whole data set. The mean correction factor was 4.80 ± 0.18 pF pl^–1 (n = 17–19 cells, 5–8 animals). G, typical experimental time course for Ca²⁺ in an experiment designed to calculate the fraction of Ca²⁺ removed from the cytosol by factors other than SERCA and NCX. Single exponentials were fit to both the caffeine‐evoked Ca²⁺ transient and the caffeine transient in the presence of 10 mm Ni⁺ (to block NCX). The decay of the caffeine‐evoked Ca²⁺ transient in the presence of Ni⁺ was expressed as a fraction of that in the absence of Ni⁺ and a value of 1 added to generate correction factors; 1.14 ± 0.03 and 1.26 ± 0.05 for young and old atrial myocytes, respectively, shown in (H) (n = 20 cells, 8–10 animals). NS, not significant. ^* P < 0.05, ^** P < 0.01.

Figure 4. Total Ca transient amplitude is unaltered with age

A, representative systolic Ca²⁺ transients converted to total Ca in young (black) and old (grey) atrial myocytes. B, mean data for total Ca transient amplitude and free Ca²⁺ transient amplitude (n = 24–33 cells, 14–19 animals). NS, not significant. ^* P < 0.05

Figure 5. Increasing Ca buffering with EGTA decreases the amplitude and rate of decay of the systolic Ca²⁺ transient in young atrial cells

A, typical Ca²⁺ transients recorded from control cells (left) and cells loaded with EGTA‐AM (right). B, mean data for Ca²⁺ transient amplitude. C, normalized Ca²⁺ transients from (A) to show the fast and slow components of decay in the presence of EGTA. The slow component represents Ca²⁺ unbinding from buffers. D, mean data for the rate of decay of the slow component. E, mean data for K _d confirming increased Ca²⁺ buffering in the presence of EGTA.

Figure 6. Peak I _Ca‐L is decreased but integrated Ca²⁺ entry maintained in old atrial myocytes

A, voltage step (upper) used to elicit I _Ca‐L (lower) in young (black) and old (grey) atrial myocytes_. B, mean data for peak I _Ca‐L (n = 47–67 cells, 19–25 animals). C, typical I _Ca‐L from young and old atrial myocytes (upper) and integrated I _Ca‐L over time (lower). Dotted line highlights the unchanged I _Ca‐L integral with age as illustrated in the mean data shown in (D). E, mean data for the rate of inactivation of I _Ca‐L fit using a double exponential with a fast and slow component presumably representing Ca²⁺ and voltage‐dependent inactivation (32–57 cells, 14–27 animals). F, amplitude of the double exponential during I _Ca‐L inactivation for both fast and slow components (32–57 cells, 14–27 animals). G, overlaid systolic Ca²⁺ transients from young (black) and old (grey) atrial myocytes. H, mean integral of the systolic Ca²⁺ transient (nmol l^–1) (29–51 cells, 15–23 animals). NS, not significant. ^* P < 0.05.

Figure 7. Key mechanisms underlying altered Ca²⁺ transient properties

Key mechanistic links defining age‐associated remodelling of the systolic Ca²⁺ transient in the sheep atria. In summary, ageing increases Ca buffering and decreases peak I _Ca‐L. Remodelling of I _Ca‐L increases SR Ca content but decreases fractional SR Ca²⁺ release. The net effect of decreased fractional release with increased SR load is maintained SR Ca release and thus unaltered total Ca transient amplitude. Increased Ca buffering results in slower and smaller Ca²⁺ transients in old atrial myocytes.