Ahnak1 abnormally localizes in muscular dystrophies and contributes to muscle vesicle release

Ahnak1 is a giant, ubiquitously expressed, plasma membrane support protein whose function in skeletal muscle is largely unknown. Therefore, we investigated whether ahnak would be influenced by alterations of the sarcolemma exemplified by dysferlin mutations known to render the sarcolemma vulnerable or by mutations in calpain3, a protease known to cleave ahnak. Human muscle biopsy specimens obtained from patients with limb girdle muscular dystrophy (LGMD) caused by mutations in dysferlin (LGMD2B) and calpain3 (LGMD2A) were investigated for ahnak expression and localization. We found that ahnak1 has lost its sarcolemmal localization in LGMD2B but not in LGMD2A. Instead ahnak1 appeared in muscle connective tissue surrounding the extracellular site of the muscle fiber in both muscular dystrophies. The entire giant ahnak1 molecule was present outside the muscle fiber and did only partially colocalize with CD45-positive immune cell infiltration and the extracelluar matrix proteins fibronectin and collagenVI. Further, vesicles shedded in response to Ca2+ by primary human myotubes were purified and their protein content was analysed. Ahnak1 was prominently present in these vesicles. Electron microscopy revealed a homogenous population of vesicles with a diameter of about 150 nm. This is the first study demonstrating vesicle release from human myotubes that may be one mechanism underlying abnormally localized ahnak1. Taken together, our results define ahnak1 in muscle connective tissue as a novel feature of two genetically distinct muscular dystrophies that might contribute to disease pathology

Potential Relevance of α1-Adrenergic Receptor Autoantibodies in Refractory Hypertension

-AAB might have a mechanistic role and could represent a therapeutic target. in cardiomyocytes and induce mesentery artery segment contraction.-AAB in hypertensive patients, and the notion of immunity as a possible cause of hypertension

Intracellular calcium in hypertrophic smooth muscle from rat urinary bladder

Objective. To explore whether infravesical outlet obstruction is associated with alterations in calcium activation of detrusor smooth muscle. Material and methods. Outlet obstruction was created by partial ligature of the urethra in female rats. Western blotting was performed using an antibody against the cytoplasmatic region of the alpha(1c) subunit of the L- type Ca2(+) channel. Intracellular calcium was measured using Fura-2 in detrusors that had been obstructed for 10 days and activated by high K+ concentrations at different extracellular Ca2(+) concentrations. The rate of force development after rapid opening of L- type Ca2(+) channels was measured in contractions initiated by flash photolysis of nifedipine in Ca2(+) containing depolarizing solution. Results. Bladder weight increased from 6293 to 254943 mg after 10 days of obstruction. Expression of the alpha(1c) subunit increased after 3 days and continued to increase until it was about fourfold greater after 10 days; however, it had not increased further at 6 weeks. This change was reversible after removal of obstruction. Activation with K+ produced a stable force at different extracellular Ca2(+) concentrations, with no difference in response between controls and rats that had been obstructed for 10 days. Intracellular Ca2(+) concentrations were lower in the obstructed group, showing that the calcium sensitivity of the contraction force had increased. The delay between the opening of L- type channels and the onset of contraction was longer in obstructed detrusors. Conclusions. Growth of detrusor muscle following obstruction is accompanied by attenuated calcium transients following activation, despite upregulation of L- type Ca2(+) channels. The Ca2(+) sensitivity of contraction was increased in obstructed detrusors. We suggest that the decreased surface: volume ratio in hypertrophic smooth muscle cells is partly involved in the lowered Ca2(+) transients. The increases in L- type calcium channels and in calcium sensitivity may be compensatory mechanisms

Modulation of muscle contraction by a cell-permeable peptide.

In contrast to immortal cell lines, primary cells are hardly susceptible to intracellular delivery methods such as transfection. In this study, we evaluated the direct delivery of several cell-permeable peptides under noninvasive conditions into living primary adult rat cardiomyocytes. We specifically monitored the functional effects of a cell-permeable peptide containing the 15 amino acid N-terminal peptide from human ventricular light chain-1 (VLC-1) on contraction and intracellular Ca2+ signals after electrical stimulation in primary adult cardiomyocytes. The transducible VLC-1 variant was taken up by cardiomyocytes within 5 min with more than 95% efficiency and localized to sarcomeric structures. Analysis of the functional effects of the cell-permeable VLC-1 revealed an enhancement of the intrinsic contractility of cardiomyocytes without affecting the intracellular Ca2+. Therefore, peptide transduction mediated by cell-penetrating peptides represents not only a unique strategy to enhance heart muscle function with no secondary effect on intracellular Ca2+ but also an invaluable tool for the modulation and manipulation of protein interactions in general and in primary cells

Agonistic antibody to the a 1 -adrenergic receptor mobilizes intracellular calcium and induces phosphorylation of a cardiac 15-kDa protein

Abstract Hypertension is a major cause for hypertrophic remodelling of the myocardium. Agonistic autoantibodies to extracellular loops of the a 1 -adrenergic receptor (a 1 -AR) have been identified in patients with arterial hypertension. However, intracellular reactions elicited by these agonistic antibodies remain elusive. An anti-peptide antibody (antia 1 ) was generated against the second extracellular loop of the a 1 -AR that bound to its peptide epitope with high affinity (K D *50 nM). We studied anti-a 1 effects on intracellular calcium (Ca i ), a key factor in cellular remodelling, and receptor-mediated cardiac protein phosphorylation. Anti-a 1 induced pronounced but transient increases in Ca i in CHO cells expressing the human a 1 -AR (CHO-a 1 ) and in neonatal cardiomyocytes. Preincubation experiments failed to demonstrate a tonic effect of anti-a 1 on Ca i . However, preincubation with the antibody attenuated the effect of the a 1 -AR antagonist prazosin. In neonatal cardiomyocytes anti-a 1 induced a robust phosphorylation of a 15-kDa protein that is involved in a 1 -AR signalling. Our data support the notion that elevation of Ca i is a general feature of agonistic antibodies' action and constitute an important pathogenic component of hypertension-associated autoantibodies. Furthermore, we suggest that agonistic antibodies to the a 1 -AR contribute to hypertrophic remodelling of cardiac myocytes, and that the cardiac 15-kDa protein is a relevant downstream target of their action

Increased expression of non-muscle myosin heavy chain-B in connective tissue cells of hypertrophic rat urinary bladder

Expression of the non-muscle myosin heavy chain-B (NM-MHC-B, also denoted as the embryonic smooth muscle myosin heavy chain, SMemb) was examined in rat urinary bladder during growth in response to a partial urinary outflow obstruction. Following obstruction, the weight of the urinary bladder increased more than five-fold within 10 days. Immunohistochemistry with a polyclonal antiserum against the C-terminal sequence of NM-MHC-B revealed very few NM-MHC-B immunoreactive cells in the control urinary bladders. In hypertrophic bladders, the number of NM-MHC-B immunoreactive cells markedly increased. The majority of such cells were found in the interstitium surrounding smooth muscle bundles and also in the subserosal and submucosal layers. Western blot analysis showed that the NM-MHC-B expression was transient; the content of NM-MHC-B immunoreactive material had doubled 10 days after obstruction and then declined towards the control level after 6 weeks. Immunohistochemistry revealed co-localization of NM-MHC-B and vimentin within the same cells. NM-MHC-B did not co-localize with smooth muscle actin, suggesting that the source of NM-MHC-B is not a de-differentiated smooth muscle cell or myofibroblast but a non-muscle cell possibly reacting to tissue distension or stress. The NM-MHC-B-positive cells could have a role in the production of extracellular matrix and growth factors or be involved in modulation of spontaneous contractile activity