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

Changes in bone architecture in <i>Lmna</i>^−/− mice.

<p>(<b>A</b>) Micro-CT analysis of total body (left panels) and femur of 4-week-old <i>Lmna</i>^−/− mice and WT littermates. Images representative of two-and three- dimensional reconstructions, obtained with a 0.9° rotation between frames on a Skyscan® 1072 instrument, are shown for WT mice (A, upper panel) and <i>Lmna</i>^−/− (A, lower panel) mice. Whole-body CT analysis of <i>Lmna</i>^−/− mice and WT littermate controls shows dramatically lower bone accumulation in <i>Lmna</i>^−/− mice with remarkable skull defects observed in the mutant mice. The right upper panels are representative of longitudinal sections. The lower right panels show the metaphysis (area just below the growth plate) (M) and distal diaphysis (cortical structure) (D). <i>Lmna</i>^−/− mice exhibited profound thinning of cortical bone, a reduction in platelike structures and a lack of trabecular connectivity. These changes correlated with von Kossa staining (<b>B</b>). Quantitation of bone parameters (<b>C</b>) further exemplified a decrease in bone volume vs. total volume (BV/TV), trabecular number (Tb.N), and cortical thickness (Ct.Th) with a concomitant increase in trabecular separation (Tb.Sp) in the mutant femora compared with the WT littermate controls. Results are expressed as the mean ± SD of eight independent analyses per group. *<i>P</i><0.001, significantly different from null mice. (<b>D</b>) Tetracycline-labeled section of the distal femur. The distance between two layers of tetracycline labels (<i>arrows</i>) visualized by epifluorescence represents bone formation that occurred during the 5-d period between tetracycline injections. Mutant mice showed a significant decrease in all parameters of bone formation including mineralized surface/bone surface (MS/BS), mineral apposition rate (MAR) and bone formation rate/bone surface (BFR/BS) as compared with their WT littermates (*<i>P</i><0.001). Photomicrographs were obtained on the Bioquant analysis system using a ×40 objective.</p

Circulating concentrations of calciotropic hormones parathyroid hormone (PTH) and 25(OH)-vitamin D [25(OH)D] in <i>Lmna</i>^−/− mice vs. WT controls.

<p>Circulating concentrations of calciotropic hormones parathyroid hormone (PTH) and 25(OH)-vitamin D [25(OH)D] in <i>Lmna</i>^−/− mice vs. WT controls.</p

Changes in osteoblast differentiation and function of 4 week-old <i>Lmna</i>^−/− mice.

<p>(<b>A and B</b>) Formation of colony forming units-osteoblasts (CFU-OB) in ex-vivo cultures of bone marrow cells from 3-week-old <i>Lmna</i>^−/− mice (lower panels) and WT controls (upper panels). The number of colony forming units-osteoblast (CFU-OB) per femur was significantly higher after 3 wks of differentiation in WT mice compared to <i>Lmna</i>^−/− mice (<b>B</b>). (<b>C</b>) Sections of plastic embedded proximal tibiae (secondary spongiosa) from <i>Lmna</i>^−/− mice and WT controls (n = 10 per group) were stained sequentially with toluidine blue (upper panels, Magnification ×40) for osteoblasts (black arrows) and osteocytes (red arrows) and ALP (lower panels, Magnification ×20) for osteoblasts (arrows). (<b>D</b>) A significant decrease (−47%) in the number of ALP expressing osteoblasts (N.Ob) and a significant decrease in osteocyte number (N.Ot) (−50%) were seen in <i>Lmna</i>^−/− mice compared with WT^+/+ mice. Micrographs are representative of those from eight different mice of each genotype. *<i>P</i><0.001. Changes in osteoblast differentiation and function correlated with changes in a serum biochemical marker of bone formation (P1NP) in <i>Lmna</i>^−/− mice compared with WT^+/+ mice. *<i>P</i><0.001. (<b>E</b>) The reduction in osteoblast differentiation in <i>Lmna</i>^−/− mice was associated with lower expression of osteocalcin (OCN) and osteopontin (OPN) at the mRNA level. There were no differences in Runx2 expression between <i>Lmna</i>^−/− mice and their WT controls. For PCR, data analysis is expressed as the ratio of the gene of interest vs. GAPDH as control. Data represent the mean±SD of triplicate determinations. *<i>P</i><0.001.</p

Mechanism of bone loss in <i>Lmna</i>^−/− mice.

<p>(<b>A and B</b>) Expression of lamin A is abolished in <i>Lmna</i>^−/− mice as compared with their WT controls. (*<i>P</i><0.001) (A). Furthermore, no difference in protein levels of Runx2 were identified in protein extracts from <i>Lmna</i>^−/− mice vs. WT control (B). Finally, expression levels of MAN-1 were significantly higher in <i>Lmna</i>^−/− mice as compared with their WT counterpart (B). Protein levels relative to tubulin were quantified by densitometry. Data represent the mean±SD of triplicate determinations. *<i>P</i><0.001. (<b>C</b>) Bone marrow cells obtained from <i>Lmna</i>^−/− mice showed a significant reduction in Runx2 DNA binding activity as compared with WT controls (*<i>P</i><0.01). The data are representative of three different experiments. (<b>D</b>) Confocal microscopy of nuclei of marrow precursors (passage 2) obtained from tibiae of <i>Lmna</i>^−/− and WT controls and cultured <i>ex vivo</i> in MSC growth media for 3 days. In cells obtained from WT mice, Runx2 (green) is widely distributed in the nucleus (upper panels, white arrows) whereas MAN-1 (red) distribution is mostly limited to the nuclear envelope. In contrast, in absence of lamin A/C (lower panels), both Runx2 and MAN1 share the same nuclear peripheral colocalization with extremely low levels of Runx2 expression (green) seen in the interior of the nuclei. Note also the smaller nuclei in MSC obtained from the mutants. DAPI (blue) was used as counterstaining only at lower magnification (left panels). In right panels, Arrows denote size of colocalization (right panels). Images are representative of cell cultures from 6 different mice.</p

Changes in osteoclast number and function of 4 week-old <i>Lmna</i>^−/− mice.

<p>(A) Sections of plastic embedded proximal tibiae from <i>Lmna</i>^−/− mice and WT (n = 10 per group) were stained sequentially for TRAP (osteoclasts, OC) (arrows, Magnification ×40). A significant decrease in the number of OC (N.Oc) showing TRAP enzyme activity (B) was seen in <i>Lmna</i>^−/− mice compared with WT controls. In addition, osteoclasts in the <i>Lmna</i>^−/− mice showed an aberrant phenotype including giant size and vacuolization (arrows). Micrographs are representative of those from eight different mice of each genotype. *<i>p</i><0.001. (<b>B</b>) Changes in bone cellularity correlated with changes in serum biochemical markers of bone resorption (CTx: C-telopeptides) in <i>Lmna</i>^−/− mice compared with WT controls. *<i>P</i><0.01.</p

Complexity of Murine Cardiomyocyte miRNA Biogenesis, Sequence Variant Expression and Function

<div><p>microRNAs (miRNAs) are critical to heart development and disease. Emerging research indicates that regulated precursor processing can give rise to an unexpected diversity of miRNA variants. We subjected small RNA from murine HL-1 cardiomyocyte cells to next generation sequencing to investigate the relevance of such diversity to cardiac biology. ∼40 million tags were mapped to known miRNA hairpin sequences as deposited in miRBase version 16, calling 403 generic miRNAs as appreciably expressed. Hairpin arm bias broadly agreed with miRBase annotation, although 44 miR* were unexpectedly abundant (>20% of tags); conversely, 33 -5p/-3p annotated hairpins were asymmetrically expressed. Overall, variability was infrequent at the 5′ start but common at the 3′ end of miRNAs (5.2% and 52.3% of tags, respectively). Nevertheless, 105 miRNAs showed marked 5′ isomiR expression (>20% of tags). Among these was miR-133a, a miRNA with important cardiac functions, and we demonstrated differential mRNA targeting by two of its prevalent 5′ isomiRs. Analyses of miRNA termini and base-pairing patterns around Drosha and Dicer cleavage regions confirmed the known bias towards uridine at the 5′ most position of miRNAs, as well as supporting the thermodynamic asymmetry rule for miRNA strand selection and a role for local structural distortions in fine tuning miRNA processing. We further recorded appreciable expression of 5 novel miR*, 38 extreme variants and 8 antisense miRNAs. Analysis of genome-mapped tags revealed 147 novel candidate miRNAs. In summary, we revealed pronounced sequence diversity among cardiomyocyte miRNAs, knowledge of which will underpin future research into the mechanisms involved in miRNA biogenesis and, importantly, cardiac function, disease and therapy.</p> </div

Novel miR*, novel non-canonical miRNAs and novel antisense miRNA.

†<p>Novel miR* that are processed within the expected window of the mature strand are labelled “generic”. Entries are ranked by tag abundance.</p>‡<p>(pre-)miRNA with known function and/or expression in the heart as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030933#pone.0030933-Matkovich1" target="_blank">[29]</a>.</p>§<p>All antisense hairpins have at least one tag aligned to the opposing side of the stem.</p>||<p>miRBase v16 annotated miRNAs removed from miRBase v17.</p>∧<p>reported in miRbase v18.</p

5′ and 3′ isomiRs in HL-1 cardiomyocytes.

<p>Thresholds were set at expression ≥150 tags (only miRNA mapped to one loci shown). Entries are ranked by tag abundance and truncated after the top 20 entries.</p><p>†(pre-)miRNA with known function and/or expression in the heart as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030933#pone.0030933-Matkovich1" target="_blank">[29]</a>.</p

miRNAs candidates deriving from novel precursors and genomic locations in HL-1 cells.

<p>(<b>A</b>) The <i>miR-30e</i> locus appears to be expressed bi-directionally, giving rise to miRNAs tags sets mapping to both, the sense (known) and antisense strands (novel; suffix –as denotes antisense-derived miRNA). (<b>B</b>) Predicted structures for both sense and antisense miR-30e hairpin precursors. (<b>C</b>) Examples of tag sets mapping to entirely new candidate miRNA loci in the murine genome. These miRNA species are tentatively named miR-N.. (N for novel). Predicted hairpin structures (RNALfold) of surrounding sequence is shown.</p