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

The Effects of Dietary Macronutrient Balance on Skin Structure in Aging Male and Female Mice

<div><p>Nutrition influences skin structure; however, a systematic investigation into how energy and macronutrients (protein, carbohydrate and fat) affects the skin has yet to be conducted. We evaluated the associations between macronutrients, energy intake and skin structure in mice fed 25 experimental diets and a control diet for 15 months using the Geometric Framework, a novel method of nutritional analysis. Skin structure was associated with the ratio of dietary macronutrients eaten, not energy intake, and the nature of the effect differed between the sexes. In males, skin structure was primarily associated with protein intake, whereas in females carbohydrate intake was the primary correlate. In both sexes, the dermis and subcutaneous fat thicknesses were inversely proportional. Subcutaneous fat thickness varied positively with fat intake, due to enlarged adipocytes rather than increased adipocyte number. We therefore demonstrated clear interactions between skin structure and macronutrient intakes, with the associations being sex-specific and dependent on dietary macronutrient balance.</p></div

Macronutrient intake influences subcutaneous fat.

<p>Response surfaces showing the association of macronutrient intake (protein, carbohydrate and fat in kJ/d) on subcutaneous adipocyte size (μm²) and adipocyte numbers (cells/10⁵μm²). (a-c) male adipocytes become grossly enlarged with high fat intake whist adipocytes proliferate with high protein intake (d-f; cells/10⁵μm²). (g-i) female adipocytes enlarge to a lesser extent than male adipocytes with high carbohydrate or fat intake and proliferate with increasing protein intake (j-i). For each 2D slice, the third factor is at its median. The red line indicates the ratio of macronutrients that minimizes each response. (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166175#pone.0166175.s005" target="_blank">S4 Table</a>)</p

The association between protein intake and male and female skin structure.

<p>H&E staining for male mouse skin layers (a-c) and female mouse skin layers (d-f), x20 magnification, scale bar = 200 μm, ‘d’ indicates area of dermis and ‘s’ indicates area of subcutaneous fat. High protein intake significantly increases male dermis thickness and thins the subcutaneous fat. In females, no effect of protein intake on skin structure was identified. Dietary composition of standard chow is protein (21%), carbohydrate (63%) and fat (16%). Mean skin thickness (a) d = 391 μm, s = 54 μm, (b) d = 275 μm s = 90 μm, (c) d = 228 μm, s = 171 μm, (d) d = 203 μm, s = 148 μm, (e) d = 127 μm, s = 233 μm, (f) d = 194 μm, s = 173 μm.</p

Dermis thickness (μm) and subcutaneous fat thickness (μm) are inversely proportional and correlate with body fat%.

<p>Dermis thickness increases with a thinner subcutaneous fat in both (a) males (R² = -0.448; P<0.001) then (b) females (R² = -0.362; P<0.001). Subcutaneous fat increases with increasing body fat % in (c) male and (d) female mice (R² = 0.549; P<0.001 and R² = 0.626; P<0.001, respectively).</p

Subcutaneous adipocytes.

<p>Representative H&E sections of mouse subcutaneous adipocytes at 40x magnification showing (a) small male adipocytes become greatly engorged with a high fat intake (b). Small female adipocyte (c) become engorged (d) but to a lesser extent than male adipocytes with a high fat diet. scale bar = 100 μm (See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166175#pone.0166175.s005" target="_blank">S4 Table</a>).</p

Macronutrients and female skin structure.

<p><b>Response surfaces showing the association between macronutrient intakes (protein, carbohydrate and fat in kJ/d) and female skin structure.</b> (a-c) epidermis thickness (μm) shows no variation with macronutrient intake. (d-f) dermis thickness (μm) increases with low carbohydrate intake. (g-i) subcutaneous fat thickness (μm) increases with high carbohydrate or high fat intake. For each 2D slice, the third factor is at its median. Red indicates maximum values, blue indicates minimum values. The red lines indicate the ratio of macronutrients that maximized each response (See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166175#pone.0166175.s004" target="_blank">S3 Table</a>).</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

RhoA mediates basal cardiac contractility in normal mice.

<p><b>A,</b> representative recordings of left ventricular pressure (LVP) and dP/dt at baseline and during saline or Y-27632 infusion (1 µM) for 5 min in NTL hearts (top panel); composite data (n = 7, bottom panel); <b>B,</b> representative Western blots (top panel) and pooled data (n = 4/group) normalized for GAPDH loading, showing p-MYPT1(Thr696), total MYPT1, and their ratio (middle panel) and p-cMLC2(Ser20), total cMLC2, and their ratio (bottom panel). Data are shown as the mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P<</i>0.001 vs. control.</p

Hypocontractility in α_1A-TG hearts is not due to heterologous desensitization but is mediated by the α_1A-AR.

<p><b>A,</b> representative recordings of left ventricular pressure (LVP) and dP/dt at baseline and during AngII infusion (100 nM) in isolated perfused contracting hearts. <b>B,</b> composite data at baseline (Control) and after AngII infusion (100 nM) for 10 min in NTL (□, n = 7) and α_1A-TG (▪, n = 9) hearts; <b>C,</b> change (Δ) from baseline for B; <b>D,</b> representative recordings of LVP and dP/dt at baseline and during α_1A-AR selective antagonist, RS100329, infusion (50 nM); <b>E,</b> composite data at baseline (Control) and after RS100329 infusion (50 nM) for 10 min in NTL (□, n = 5) and α_1A-TG (▪, n = 4) hearts. Data are shown as the mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p