Plasma membrane Ca ²⁺ ‐ATPase 1 is required for maintaining atrial Ca ²⁺ homeostasis and electrophysiological stability in the mouse

Figure 1. Expression of PMCA1 is significantly reduced in the atria of PMCA1cko mice compared to PMCA1loxP/loxP controls A, a representative western blot image of PMCA1 protein expression in atrial extracts. α‐Tubulin was used as a loading control. B, the level of expression was quantified showing a significant reduction in PMCA1/α‐tubulin ratio in PMCA1cko mice atria (0.158 ± 0.073/0.017 ± 0.016, P = 0.026, 3 replicates from 3 animals each). C, the efficiency of the PMCA1 knockout in PMCA1cko mouse atria was further confirmed by immunohistochemical staining of atrial sections compared with PMCA1loxP/loxP mice. D, the mRNA levels of PMCA4 in both atria and ventricle remained unchanged in PMCA1loxP/loxP and PMCA1cko mice. E, the in vivo heart rates of PMCA1loxP/loxP and PMCA1cko mice showed no significant difference with either 1‐week isoprenaline treatment or acute dobutamine injection treatment. The bars show the mean + SD.

Figure 2. Ex vivo Langendorff recordings of ECG and atrial MAP of PMCA1cko and PMCA1loxP/loxP hearts under baseline condition at 1 mm [Ca2+]o A, representative traces of the ex vivo Langendorff ECG (left) and MAP (right) recordings. There was no significant difference in PR, QRS, QTc and RR intervals (data not shown). Two PES protocols, S1S2 and S1S1 pacing, were introduced to the atrial septum to stress the hearts. B, PMCA1cko hearts exhibited frequent short AT/AF when PES pacing was applied while none of the PMCA1loxP/loxP hearts exhibited any arrhythmic events in ECG (left panel) and MAP (right panel) recordings. C, representative traces of ECG (upper panel) and MAP (lower panel) recordings under S1S1 PES pacing. PMCA1cko hearts showed sustained or non‐sustained AT/AF when PES pacing was applied while none of the PMCA1loxP/loxP hearts exhibited any arrhythmic events. D, the percentage occurrence of arrhythmic events as a result of different S1S1 PES pacing frequencies in PMCA1cko and PMCA1loxP/loxP hearts. E, the action potential duration at 90% (APD90) and effective refractory period (ERP) under baseline and series of S1S1 PES pacing (90, 70, 50 and 30 ms) showed no significant…

Figure 3. The ex vivo Langendorff recordings of ECG and MAP of PMCA1cko and PMCA1loxP/loxP hearts at 2 mm [Ca2+]o A, representative traces of the ex vivo Langendorff ECG (left) and MAP (right) recordings. There was no significant difference in PR, QRS, QTc and RR intervals (data not shown). B, PMCA1cko hearts exhibit frequent short AT/AF when PES pacing was applied whilst none of the PMCA1loxP/loxP hearts exhibited any arrhythmic events of ECG (left panel) and MAP (right panel) recordings. C, representative traces of ECG (upper panel) and MAP (lower panel) recordings under S1S1 PES pacing. PMCA1cko hearts showed sustained or non‐sustained AT/AF when PES pacing was applied while none of the PMCA1loxP/loxP hearts exhibited any arrhythmic events. D, the percentage occurrence of arrhythmic events as a result of different S1S1 PES pacing frequencies in PMCA1cko and PMCA1loxP/loxP hearts. E, the APD90 and ERP under baseline and series of S1S1 PES pacing (90, 70, 50 and 30 ms) showed no significant differences in PMCA1loxP/loxP and PMCA1cko mice under 2 mm [Ca2+]o conditions.

Figure 4. The occurrences of arrhythmic type of AP and calcium sparks in isolated PMCA1cko and PMCA1loxP/loxP atrial cells at 1 mm [Ca2+]o and 2 mm [Ca2+]o A and B, the characteristics of AP and corresponding calcium transient were analysed for PMCA1cko (grey trace) mice and PMCA1loxP/loxP controls (black trace) at 1 mm [Ca2+]o (A) or 2 mm [Ca2+]o (B) with 1 and 3 Hz stimulation. P indicates the stimulation pulse. C, there was an increase in arrhythmic type of AP occurrence in PMCA1cko mice with faster stimulation frequency under 2 mm [Ca2+]o. D and E, the number of calcium sparks measured by linescan was higher in PMCA1cko myocytes under both baseline (D) and 2 mm [Ca2+]o (E) conditions. F and G, this increase in the number of calcium sparks was also demonstrated in PMCA1cko myocytes measured with a spinning‐disc microscope under both baseline (F) and 2 mm [Ca2+]o (G) conditions. H, the quantification of the number of calcium sparks per cell per second.

Figure 5. Calcium extrusion via NCX and SR content were examined in PMCA1cko mice and PMCA1loxP/loxP controls at 1 mm [Ca2+]o (A) or 2 mm [Ca2+]o (B) conditions Representative traces of integral NCX, caffeine‐induced current and corresponding calcium transient are shown under baseline (Aa, c and d) and 2 mm [Ca2+]o (Ba, c and d) conditions. The time decay of I NCX in PMCA1cko myocytes was significantly prolonged under both baseline (Af) and 2 mm [Ca2+]o (Bf) conditions. The time decay of corresponding calcium transient in PMCA1cko myocytes showed significantly longer duration than PMCA1loxP/loxP myocytes under 2 mm [Ca2+]o (Bh) but not under baseline (Ah) conditions. There was no significant difference between the SR content of PMCA1loxP/loxP and PMCA1cko mice under baseline (Ab) and 2 mm [Ca2+]o (Bb) conditions. The density of I NCX and corresponding calcium transient amplitude showed no significant difference between the SR content of PMCA1loxP/loxP and PMCA1cko mice under baseline (Ae and g) and 2 mm [Ca2+]o (Be and g) conditions. Representative traces of I LTCC are shown under baseline (Ai) and 2 mm [Ca2+]o (Bi) conditions. The peak amplitude of I LTCC in PMCA1cko myocytes and…