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

<i>Tgm2</i> deletion has no effect on insulin secretion by islets.

<p>Islets were isolated (A) from 6 month-old male B6 WT and TG2^−/− (i) or 129 WT and TG2^−/− (ii) mice fed a normal chow diet (chow) or fed a normal chow diet for 3 months followed by a high-fat diet for 3 months (fat) or (B) from 3 month old male 129 WT and TG2^−/− mice fed a normal chow diet. (A) Isolated islets, 5 per group (n = 7), were incubated in Krebs-Ringer bicarbonate (KRB) buffer containing 2.8 mM glucose or 16.8 mM glucose or 16.8 mM glucose plus 0.1 mM carbachol, 37°C, 1 h. (B) Groups of 70 islets (n = 3 for WT and n = 2 for TG2^−/−) were perifused in KRB containing 2.8 mM glucose for 10 min before a stimulatory period of 60 min with KRB containing 16.8 mM glucose. The perifusate was switched back to 2.8 mM glucose for 30 min to allow insulin secretion to return to basal levels before exposure to 16.8 mM glucose and 100 µM carbachol for 30 min. The perifusate was switched back to 2.8 mM glucose for 10 min before non-metabolic depolarisation with 24 mM KCl to activate voltage-gated Ca²⁺ channel-triggered insulin secretion. One min fractions of the perifusate were collected for the first 10 min, then 2 min fractions were collected at a flow rate of 0.5 ml/min. Insulin content of supernatant and of islets was determined by radioimmunoassay. Data are presented as means ± SEM. Overall <i>P</i> values calculated using 2-way ANOVA are shown at the top; individual Bonferroni <i>post hoc</i> test results are shown above the columns.**<i>P</i><0.01; ***<i>P</i><0.001.</p

Neither <i>Tgm2</i> deletion nor substitution with <i>Tgm2^R579A</i> affects glucose homeostasis in 3 month-old B6 mice.

<p>(A, B) Fasted B6 WT and TG2^R579A/R579A mice were subjected to intraperitoneal glucose tolerance tests or (C, D) fed B6 WT and TG2^−/− or B6 WT and TG2^R579A/R579A mice were subjected to intraperitoneal insulin tolerance tests, and blood glucose (A, C, D) or serum insulin levels (B) were determined. Data are presented as means ± SEM (n = 9–11). Overall <i>P</i> values calculated using 2-way ANOVA are shown at the top; individual Bonferroni <i>post hoc</i> test results are shown above the line profiles. **<i>P</i><0.01; ***<i>P</i><0.001.</p

<i>Tgm2</i> deletion has no effect on oral glucose tolerance tests in 6 month-old male mice.

<p>Male B6 WT and TG2^−/− or 129 WT and TG2^−/− mice (3 months old raised on a normal chow diet) were fed normal chow for 3 months (A, C) or fed a high-fat diet for 3 months (B, D). Fasted male mice were then subjected to oral glucose tolerance tests, and blood glucose (A, B) and serum insulin (C, D) levels were determined. Data are presented as means ± SEM (n = 7). Overall <i>P</i> values calculated using 2-way ANOVA are shown at the top; individual Bonferroni <i>post hoc</i> test results are shown above the line profiles. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001.</p

Contractility in α_1A-TG isolated working hearts.

<p><b>A,</b> baseline left ventricular systolic pressure (LVSP), dP/dt_max and dP/dt_min of isolated perfused contracting NTL (n = 17) and α_1A-TG (n = 24) hearts. <b>B,</b> representative recordings of left ventricular pressure (LVP) and dP/dt at baseline and during A61603 infusion (100 pM). <b>C,</b> composite data obtained from NTL (◊, n = 6) and α_1A-TG (•, n = 7) hearts at baseline (C) and dose-response to A61603. Data are shown as the mean ± SEM. *<i>P</i><0.05, *<i>*P<</i>0.01; ***P<0.001 vs. NTL.</p

Randomly fed body weights (g) and blood glucose (mM) concentrations in 3–4 month-old mice.

<p>Values are presented as means ± SEM (n = 9–11).</p>*<p><i>P</i><0.001 vs male.</p

Fasting body weights (g), blood glucose (mM), serum insulin (ng/ml) and total insulin content per islet (ng/islet) in 6-month old mice fed a normal chow diet (Chow diet) or fed a normal chow diet for 3 months followed by a high-fat diet for 3 months (High-fat diet).

<p>Values are presented as means ± SEM (n = 7).</p>x<p><i>P</i><0.05 vs chow diet;</p>*<p><i>P</i><0.001 vs chow diet;</p>#<p><i>P</i><0.001 vs WT;</p>†<p><i>P</i><0.05 vs corresponding B6 genotype;</p>‡<p><i>P</i><0.001 vs corresponding B6 genotype.</p

Mechanism of α_1A-TG hypocontractility.

<p>Western blot analyses of myofilament proteins and RhoA activity in NTL (□) and α_1A-TG (▪) hearts after infusion of saline or RS100329 (50 nM) for 8 min. In each panel, representative Western blots and pooled data (n = 3/group) are shown: <b>A,</b> p-cMLC2(Ser20), total cMLC2, and their ratio; <b>B,</b> p-MYPT1(Thr696), total MYPT1, and their ratio; <b>C,</b> RhoA protein expression, RhoA activity and the relationship between dP/dt_max and RhoA activity, where data are shown from NTL isolated hearts treated with saline (○) or RS100329 (•) and α_1A-TG hearts treated with saline (Δ) or RS100329 (▴). Western blot data are normalized to GAPDH expression. Data are shown as the mean ± SEM. <i>*P<</i>0.05, *<i>*P<</i>0.01.</p

Generation of coisogenic B6 <i>Tgm2^R579A</i> knock-in mice constitutively active for transamidating activity.

<p>A. Gene targeting strategy. i. Original locus showing location of Arg579 and <i>Bgl</i>II site (red line) in exon 11 (black box) of <i>Tgm2</i>. PCR primers, FP1 & RP2, yield a 250 bp <i>Bgl</i>II-cleavable fragment. ii. Targetted locus showing <i>Tgm2^R579A</i> mutation, introduced <i>Nhe</i>I site, and insertion of a LoxP-flanked PGK–neomycin resistance selection cassette between exons 11 & 12. iii. Cre-deleted locus showing <i>Tgm2^R579A</i> mutation, introduced <i>Nhe</i>I site, and remnant loxP site. PCR primers, FP1 & RP2, yield a 320 bp <i>Nhe</i>I-cleavable fragment. B. PCR primers, FP1 and RP2, distinguish the 250 bp wild-type <i>Tgm2</i> allele from the 320 bp <i>Tgm2^R579A</i> knock-in allele. C. Homogenized tissues from WT or TG2^R579A/R579A mice (n = 3) were size-fractionated and TG2 levels detected by Western blot were normalized for loading using anti-GAPDH for heart, liver, MEF or tubulin for islets. D. GTPγS inhibition of <i>in vitro</i> calcium-activated transamidase activity was assayed in WT or TG2^R579A/R579A liver lysates (n = 3) as described in “Materials and Methods”. Transamidase activity was maximal (100%) with addition of 2mM CaCl₂. <i>P</i> values were calculated using 1-way ANOVA. E. Intracellular transamidase activity was assayed in intact WT or TG2^R579A/R579A murine embryonic fibroblasts (n = 6) as described in “Materials and Methods”. <i>P</i> values were calculated using 2-way ANOVA with the Bonferroni <i>post hoc</i> test. Data are presented as means ± SEM. **<i>P</i><0.01; ***<i>P</i><0.001.</p

Contractility in α_1A-TG cardiomyocytes.

<p>Indices of excitation-contraction coupling before and after α_1A-AR agonist stimulation with phenylephrine (PE) in NTL (◊, n = 7) and α_1A-TG (•, n = 7) CMs. <b>A,</b> basal [Ca²⁺]_I; <b>B,</b> amplitude of the systolic [Ca²⁺]_i rise (Peak-Basal); <b>C,</b> percent cell shortening. Data are shown as the mean ± SEM. <i>*P<</i>0.05 vs. NTL.</p