Force development and intracellular Ca2+ in intact cardiac muscles from gravin mutant mice

Abstract

Gravin (AKAP12) is an A-kinase-anchoring-protein that scaffolds protein kinase A (PKA), β₂-adrenergic receptor (β₂-AR), protein phosphatase 2B and protein kinase C. Gravin facilitates β₂-AR-dependent signal transduction through PKA to modulate cardiac excitation-contraction coupling and its removal positively affects cardiac contraction. Trabeculae from the right ventricles of gravin mutant (gravin-t/t) mice were employed for force determination. Simultaneously, corresponding intracellular Ca²⁺ transient ([Ca²⁺]_i) were measured. Twitch force (T_f)-interval relationship, [Ca²⁺]_i-interval relationship, and the rate of decay of post-extrasysolic potentiation (R_f) were also obtained. Western blot analysis were performed to correlate sarcomeric protein expression with alterations in calcium cycling between the WT and gravin-t/t hearts. Gravin-t/t muscles had similar developed force compared to WT muscles despite having lower [Ca²⁺]_i at any given external Ca²⁺ concentration ([Ca²⁺]_o). The time to peak force and peak [Ca²⁺]_i were slower and the time to 75% relaxation was significantly prolonged in gravin-t/t muscles. Both T_f-interval and [Ca²⁺]_i-interval relations were depressed in gravin-t/t muscles. R_f, however, did not change. Furthermore, Western blot analysis revealed decreased ryanodine receptor (RyR2) phosphorylation in gravin-t/t hearts. Gravin-t/t cardiac muscle exhibits increased force development in responsiveness to Ca²⁺. The Ca²⁺ cycling across the SR appears to be unaltered in gravin-t/t muscle. Our study suggests that gravin is an important component of cardiac contraction regulation via increasing myofilament sensitivity to calcium. Further elucidation of the mechanism can provide insights to role of gravin if any in the pathophysiology of impaired contractility.

Keywords: Cardiac muscle; Contraction; Force development; Gravin; Intracellular calcium; Trabeculae.

Fig. 1

Raw tracings of stress development (left) and [Ca²⁺]_i transient (right) from WT (upper panels) and gravin-t/t (lower panels) trabecular muscles at two [Ca²⁺]_o (2 and 5 mM). Note that [Ca²⁺]_i transients were lower in gravin-t/t muscles while stress remained unchanged.

Fig. 2

Pooled data of stress (A) and [Ca²⁺]_i transients (B) at varied [Ca²⁺]_o (1-5 mM) from WT and gravin-t/t muscles. Both stress and [Ca²⁺]_i transient increased as [Ca²⁺]_o was raised. Stress was similar in both groups of muscles but [Ca²⁺]_i transients remained significantly lower in gravin-t/t muscles (P<0.001; repeated MANOVA, n=8 in each group).

Fig. 3

Dynamics of stress and [Ca²⁺]_i transient of trabecular muscles from WT and gravin-t/t mice. The time to peak stress (A) and [Ca²⁺]_i (B) was prolonged in gravin-t/t muscles (as determined by MANOVA) whereas the time to 50% relaxation of stress (C) and [Ca²⁺]_i (D) was not different between WT and gravin-t/t muscles. However, the initial relaxation time (time to 75% of peak) in twitch (E) was significantly slowed in gravin-t/t muscles (P<0.01 repeated MANOVA) whereas the initial relaxation time (time to 75% of peak) in [Ca²⁺]_i (F) was not different between WT and gravin-t/t muscle. (n=8 in each group)

Fig. 4

(A) Peak stress and peak [Ca²⁺]_i transient relationships of trabecular muscles from WT and gravin-t/t mice (pooled data, n=8 in each group). In WT, force = 41 [Ca²⁺]_i – 8.22; in gravin-t/t, force = 67 [Ca²⁺]_i – 7.08. The relationship is also shifted leftward in gravin-t/t muscles. These two relationships are significantly different (P<0.001; MANOVA). (B) Representative phase-plane plots for stress vs. [Ca²⁺]_i transient from WT and gravin-t/t muscles. The phase-plane plot is stress vs. [Ca²⁺]_i transient point-to-point over the entire trajectory of the twitch. Note the phase-plane plot of gravin-t/t muscle is shifted to the left. (C) Comparison of [Ca²⁺]_i at which stress relaxed to 50%. *P<0.05 vs. WT; n=6, [Ca²⁺]_o = 1.0 mM.

Fig. 5

Stress-interval (A) and [Ca²⁺]_i transient-interval (B) relations of trabecular muscles from WT and gravin-t/t mice. The test (rest) intervals are shown on logarithmic scale for the sake of clarity. At short test intervals (<5 s), stress recovered quickly, followed by a slower rise in both groups of muscles. At intervals greater than 60 s, stress started to decline. Gravin-t/t muscle also had lower stress at rest intervals greater than 10 s. [Ca²⁺]_i transients exhibited similar pattern as test interval changed (right), with [Ca²⁺]_i transient being lower in gravin-t/t muscles. [Ca²⁺]_o = 1.0 mM. Note that rate of recovery of [Ca²⁺]_i transient is similar at shorter test intervals in both groups. But the increase (or potentiation) is much slower at test intervals great than 10 s and the total increase is also lower in gravin-t/t muscles. See text for detailed discussion. [Ca²⁺]_o = 1.0 mM.

Fig. 6

Recirculation fraction (R_f) of Ca²⁺ in WT (n=5) and gravin-t/t muscles (n=7). R_f was determined from the slopes of the linear relationships between consecutive twitches and their corresponding [Ca²⁺]_i transients at a baseline stimulation rate of 0.5 Hz after potentiation of both stress development and [Ca²⁺]_i transient. Stress _(n) and Stress _(n+1) are two consecutive twitches, and [Ca²⁺]_{i (n)} and [Ca²⁺]_{i (n+1)} refer two consecutive [Ca²⁺]_i transients. [Ca²⁺]_o = 1.0 mM.

Fig. 7

Ca²⁺-related protein expression. Western blot analysis of (A) sarcoplasmic reticulum Ca²⁺-ATPase (SERCA), (B) protein phosphatase-1 inhibitor-1 (IPP-1) following acute vehicle (ascorbic acid; 0.002%) or ISO infusion (10 μg/g/min), (C) L-type calcium channel (CP α1C), (D) ryanodine receptor (RyR2), (E) calsequestrin (CSQ2), and (F) sodium / calcium exchanger (NCX) in heart homogenates from WT and gravin-t/t mice. (Lane 1: WT (vehicle); Lane 2: WT (ISO); Lane 3: gravin-t/t (vehicle); Lane 4: gravin-t/t (ISO)). The bar graphs show the ratio of phosphorylated (phospho) to total protein or target protein to GAPDH. All lanes of a specific protein were detected on the same blot and a vertical line indicates where the blots are not contiguous. Data are expressed as the mean ± S.E.M.; n = 4 to 6 samples; *P<0.05 vs. baseline of same phenotype.