Kontracepcija

<div><p>Aims</p><p>To determine the mechanisms by which the α_1A-adrenergic receptor (AR) regulates cardiac contractility.</p><p>Background</p><p>We reported previously that transgenic mice with cardiac-restricted α_1A-AR overexpression (α_1A-TG) exhibit enhanced contractility but not hypertrophy, despite evidence implicating this Gα_q/11-coupled receptor in hypertrophy.</p><p>Methods</p><p>Contractility, calcium (Ca²⁺) kinetics and sensitivity, and contractile proteins were examined in cardiomyocytes, isolated hearts and skinned fibers from α_1A-TG mice (170-fold overexpression) and their non-TG littermates (NTL) before and after α_1A-AR agonist stimulation and blockade, angiotensin II (AngII), and Rho kinase (ROCK) inhibition.</p><p>Results</p><p>Hypercontractility without hypertrophy with α_1A-AR overexpression is shown to result from increased intracellular Ca²⁺ release in response to agonist, augmenting the systolic amplitude of the intracellular Ca²⁺ concentration [Ca²⁺]_i transient without changing resting [Ca²⁺]_i. In the <i>absence</i> of agonist, however, α_1A-AR overexpression <i>reduced</i> contractility despite unchanged [Ca²⁺]_i. This hypocontractility is not due to heterologous desensitization: the contractile response to AngII, acting via its Gα_q/11-coupled receptor, was unaltered. Rather, the hypocontractility is a pleiotropic signaling effect of the α_1A-AR in the absence of agonist, inhibiting RhoA/ROCK activity, resulting in hypophosphorylation of both myosin phosphatase targeting subunit 1 (MYPT1) and cardiac myosin light chain 2 (cMLC2), reducing the Ca²⁺ sensitivity of the contractile machinery: all these effects were rapidly reversed by selective α_1A-AR blockade. Critically, ROCK inhibition in normal hearts of NTLs without α_1A-AR overexpression caused hypophosphorylation of both MYPT1 and cMLC2, and rapidly reduced basal contractility.</p><p>Conclusions</p><p>We report for the first time pleiotropic α_1A-AR signaling and the physiological role of RhoA/ROCK signaling in maintaining contractility in the normal heart.</p></div

Fatigue.

<p>The time course of force decline during 30 seconds of fatiguing stimulation is shown in (A) for males and (B) for females. It can be seen that in both males and females, EDL muscles from aged animals fatigued less rapidly than muscles from adult animals. At the end of the 30-second fatigue protocol, muscles from aged animals were able to generate a significantly higher percentage of their pre-fatigue force than muscles from adult animals. (Error bars are within thickness of symbols.)</p

Force-frequency curves.

<p>Force-frequency curves for EDL muscles from adult and aged animals are shown in (A) for males and (B) for females. In males, the force-frequency curve in aged animals is shifted upwards at low frequencies, reflecting a significantly higher twitch-to-tetanus ratio than in adult animals. In females, the force-frequency curve for aged mice is shifted leftwards, reflecting a significantly lower half-frequency than in adult animals.</p

Tetanus relaxation.

<p>(A) shows force recordings of the tetanus for two EDL muscles in our sample. It shows the final stages of the period of stimulation, and the initial stages of relaxation. It can be seen that force declines linearly in the initial stages of relaxation. In the muscle that relaxes more slowly (dashed line), this linear phase has a longer duration and a reduced steepness of slope compared with the faster-relaxing muscle (full line). We examined this linear phase both before and after subjecting the muscles to a fatiguing stimulation protocol. The duration of the linear phase is shown in (B) and the steepness of the slope of this linear phase is shown in (C).</p

Maximum forces.

<p>In females, EDL muscles of aged animals had lower absolute force (A) than muscles from adult animals. Specific force (B) was lower in aged compared with adult animals, in both males and females.</p

Analysis of ramp phase of eccentric contractions.

<p>The diagram in (A) shows the change in force (full line) and length (dotted line) as the muscle is stretched during an eccentric contraction. The dashed line is the slope (first derivative) of the force-time curve. The plot of slope exhibits three distinct phases, and each phase is separated by a transition point (T₁ and T₂). For each muscle, we measured the force and length at T₁ and T₂. The results are shown in the graphs on the right. There were no significant differences between adult and aged animals in either the force (B) or length (C) at which the transition points occurred.</p

Mass and cross-sectional area.

<p>In males, EDL muscles of aged animals had higher mass (A) and cross-sectional area (B) than muscles from adult animals. In females, there were no significant differences between adult and aged animals.</p

Eccentric contractions and stiffness.

<p>In a separate group of mice, EDL muscles were subjected to a mild eccentric contraction protocol. (A) is an example of an eccentric contraction, showing the change in force and length as the muscle is stretched by 15% of its optimal length L₀, then returned to resting length. 3 such eccentric contractions were performed for each muscle. (B) shows the force deficits following these eccentric contractions. Force deficits in aged mice were significantly higher than in adult mice. As an indicator of muscle stiffness, we also measured the percentage change in force for every 1% change in length during the stretch phase of the eccentric contraction. The results are shown in (C). The change in force was significantly higher in aged than in adult mice, indicative of greater stiffness in the muscles of aged mice.</p

Twitch parameters.

<p>Time-to-peak (A) was longer in EDL muscles of aged male mice than in adult male mice. There were no differences in twitch half-relaxation time (B) between aged and adult mice, in either males or females.</p

Twitch and tetanus in EDL muscles before and after creatine treatment.

<p><i>A</i>, twitch to tetanus ratios expressed relative to the pre-fatigue twitch to tetanus ratio. Before creatine incubation, the twitch to tetanus ratio in fatigued muscle was similar to that in the pre-fatigue state. After creatine incubation however, the twitch to tetanus ratio in the fatigued state was significantly higher than in the pre-fatigue state. Statistically significant differences are indicated by “*” (<i>P</i><0.05). <i>B</i>, force tracings of twitches and 100-Hz tetani obtained from one EDL muscle. In the fatigued muscle, creatine incubation increases the twitch force and tetanic force. In the recovered muscle however, creatine incubation makes little difference to the twitch and tetanic forces.</p