Contractile Behavior of Mouse Aorta Depends on SERCA2 Isoform Distribution: Effects of Replacing SERCA2a by SERCA2b

FIGURE 1. SERCA2 expression in the aorta of SERCA2a/b and SERCA2b/b mice. (A) Real-time RT-PCR on RNA isolated from aortic segments of SERCA2a/b mice and SERCA2b/b mice. SERCA2a and SERCA2b transcripts were determined versus the reference gene transcript β-actin (ACTB) (n = 3). (B) Immunofluorescent staining of sections of thoracic aorta (Ao) of SERCA2a/b (upper row) and SERCA2b/b (lower row) mice (n = 3) with antibodies against SERCA2a (left column) and SERCA2b (right column). For SERCA2a, sections of left heart ventricle (H) are included. (Lu) indicates lumen of the aorta. SERCA2 was stained green, whereas the nuclei were stained blue with DAPI. A 10 times larger image of the aortic wall for SERCA2a is also shown for the SERCA2a/b mouse aorta (insert) with the arrow indicating SERCA2b staining of ECs. Horizontal scale bar is 320 μm (30 μm for the insert). (C) Representative examples of Western blot for the SERCA2a isoform (left, n = 4), SERCA2b isoform (middle, n = 5) and total SERCA (right, n = 3) in aortic segments of SERCA2a/b (S2a/b) and SERCA2b/b (S2b/b) mice (last two lanes) with the respective peptides as positive controls (SERCA2a and SERCA2b, first two lanes). (D) Summary of the…

FIGURE 2. Intracellular Ca2+ signals and concomitant force development induced by stepwise increase of K+ in aortic segments of SERCA2a/b and SERCA2b/b mice. Sensitivity of SERCA2a/b (circles) and SERCA2b/b (squares) aortic segments to depolarization with elevated external K+. (A) Ca2+ signals and concomitant force development with increasing concentrations of extracellular K+, measured in Fura-2-loaded endothelium-denuded aortic segments mounted in a myograph (n = 4). Force at increasing K+ for endothelium-intact aortic segments measured in an isometric organ bath in control (B) and after inhibition of eNOS with 300 μM L-NAME (C) (B,C: n = 5).

FIGURE 3. Isometric contractions by PE are higher in aortic segments of SERCA2b/b compared to SERCA2a/b. Concentration-effect curves (A) and respective logEC50 values (B) for contractions by PE (10– 9 to 3*10– 5 M) in the absence (open symbols) and presence (closed symbols) of 300 μM L-NAME in SERCA2a (circles) and SERCA2b (squares) aortic segments. Graphs show the contraction induced by 1 μM PE in the absence (C) and presence (D) of 1 μM levcromakalim. 10 Min after the addition of PE, 300 μM L-NAME was added in all conditions to inhibit release of basal NO. *, **p < 0.05, 0.01 SERCA2b/b versus SERCA2a/b (n = 5); #, ###p < 0.05, 0.001 + L-NAME versus –L-NAME.

FIGURE 4. Phasic contractions by PE (1 μM during 0 mM Ca2+ challenge) display slower and smaller spontaneous SR contractile Ca2+ release but always faster relaxation kinetics in SERCA2b/b segments. (A) Phasic contractions by PE (1 μM) upon 0 mM Ca2+ challenge of various duration decrease with duration of the 0 mM Ca2+ challenge, but at each time interval display consistently faster relaxation kinetics in SERCA2b/b than in SERCA2a/b segments. (B) The AUC of the phasic contraction, representative for the SR Ca2+ store content, is significantly lower, but more complete in SERCA2b/b segments. (C) The decline of the AUC of the phasic contraction over different durations of 0 mM Ca2+ challenges tended to be slower in SERCA2b/b segments. *p < 0.05, SERCA2b/b (S2b/b) versus SERCA2a/b (S2a/b), (n = 4–5).

FIGURE 5. Tonic contractions by PE (upon re-addition of 3.5 mM Ca2+) in SERCA2a/b and SERCA2b/b segments. (A) Following the phasic contractions, tonic contractions by PE (1 μM) were determined in SERCA2a/b and SERCA2b/b mouse aortic segments by re-addition of 3.5 mM Ca2+. Tonic contractions by PE tended to be higher in SERCA2b/b than in SERCA2a/b segments. (B) In the presence of 35 μM diltiazem tonic contractions by PE were higher in SERCA2b/b than in SERCA2a/b segments. (C) Amplitudes of contractions after 250 s in the absence and presence of diltiazem. **, ###p < 0.01, 0.001 SERCA2b/b versus SERCA2a/b, ###: diltiazem versus control (n = 5).

FIGURE 6. Amount of re-filling of the PE-sensitive contractile SR Ca2+ stores in SERCA2a/b and SERCA2b/b segments. (A) Experimental protocol to investigate PE (1 μM)-sensitive phasic contractions in 0 mM Ca2+ after a period of 5 min of Ca2+ re-addition (+ Ca2+) to refill the SR Ca2+ stores. (B) Phasic contractions of the first (PE1) and second (PE2) phasic contraction by 1 μM PE in 0 mM Ca2+ in SERCA2a/b (black) and SERCA2b/b (blue) aortic segments. (C) Ratio of AUC of the first and second PE application (PE1/PE2 in%) in both strains. When after PE1 35 μM diltiazem was added, refilling was attenuated in SERCA2a/b segments (D), but was less affected in SERCA2b/b segments (E). (F) Ratio of AUC of the first and second PE application (PE1/PE2 in%) in control (open bars) and after addition of 35 μM diltiazem (patterned bars) in both strains. *, **, ***p < 0.05, 0.01, 0.001 SERCA2b/b versus SERCA2a/b (n = 5), ##p < 0.01 diltiazem versus control (n = 5).

FIGURE 7. SERCA Ca2+ re-uptake inhibition with CPA caused similar intracellular Ca2+ signals, but increased isometric force signals in SERCA2b/b more than SERCA2a/b aortic segments. (A) Intracellular Ca2+ and (B) isometric force were simultaneously measured in endothelium-denuded (1 mm width) aortic segments (n = 5) of SERCA2a/b and SERCA2b/b mice when treated with 10 μM CPA as indicated by the horizontal bar. *, ***p < 0.05, 0.001 SERCA2b/b versus SERCA2a/b. (C,D) Tonic contractions by 10 μM CPA were measured in (2 mm width) aorta segments treated with 300 μM L-NAME to inhibit eNOS production. CPA-induced contractions were increased in SERCA2b/b also after inhibition of L-type Ca2+ influx with diltiazem. CPA was added during a 0 mM Ca2+ challenge, while re-addition of 3.5 mM Ca2+ (+ CPA solution) resulted in a contractile response, both in the absence and presence of 35 μM diltiazem. *, **, ***p < 0.05, 0.01, 0.001 SERCA2b/b versus SERCA2a/b, #, ##p < 0.05, 0.01 diltiazem versus control (n = 5).

FIGURE 8. Intracellular Ca2+ signals and concomitant force development induced by CPA in endothelium-intact aortic segments of SERCA2a/b and SERCA2b/b mice stimulated with 1 μM PE. (A,B) Representative example of intracellular Ca2+ (upper row) and concomitant force (lower row) upon addition of 1 μM PE (down triangle), CPA (10–8–10–5 M, circles) and wash (up triangle) in a SERCA2a/b segment (black, A) and SERCA2b/b segment (blue, B). (C) % increase of Ca2+ (top) and % relaxation as a function of [CPA].