Mechthild M. Schroeter

Developmental changes in contractility and sarcomeric proteins from the early embryonic to the adult stage in the mouse heart

Developmental changes in force‐generating capacity and Ca2+ sensitivity of contraction in murine hearts were correlated with changes in myosin heavy chain (MHC) and troponin (Tn) isoform expression, using Triton‐skinned fibres. The maximum Ca2+‐activated isometric force normalized to the cross‐sectional area (FCSA) increased mainly during embryogenesis and continued to increase at a slower rate until adulthood. During prenatal development, FCSA increased about 5‐fold from embryonic day (E)10.5 to E19.5, while the amount of MHC normalized to the amount of total protein remained constant (from E13.5 to E19.5). This suggests that the development of structural organization of the myofilaments during the embryonic and the fetal period may play an important role for the improvement of force generation. There was an overall decrease of 0.5 pCa units in the Ca2+ sensitivity of force generation from E13.5 to the adult, of which the main decrease (0.3 pCa units) occurred within a short time interval, between E19.5 and 7 days after birth (7 days pn). Densitometric analysis of SDS‐PAGE and Western blots revealed that the major switches between troponin T (TnT) isoforms occur before E16.5, whereas the transition points of slow skeletal troponin I (ssTnI) to cardiac TnI (cTnI) and of β‐MHC to α‐MHC both occur around birth, in temporal correlation with the main decrease in Ca2+ sensitivity. To test whether the changes in Ca2+ sensitivity are solely based on Tn, the native Tn complex was replaced in fibres from E19.5 and adult hearts with fast skeletal Tn complex (fsTn) purified from rabbit skeletal muscle. The difference in pre‐replacement values of pCa50 (−log([Ca2+]m−1)) required for half‐maximum force development) between E19.5 (6.05 ± 0.01) and adult fibres (5.64 ± 0.04) was fully abolished after replacement with the exogenous skeletal Tn complex (pCa50= 6.12 ± 0.05 for both stages). This suggests that the major developmental changes in Ca2+ sensitivity of skinned murine myocardium originate primarily from the switch of ssTnI to cTnI.

Inhibition of contraction and myosin light chain phosphorylation in guinea-pig smooth muscle by p21-activated kinase 1

The p21‐activated protein kinases (PAKs) have been implicated in cytoskeletal rearrangements and modulation of non‐muscle contractility. Little, however, is known about the role of the PAK family members in smooth muscle contraction. Therefore, we investigated the effect of the predominant isoform in vascular smooth muscle cells, PAK1, on contraction and phosphorylation of the regulatory light chains of myosin (r‐MLC) in Triton‐skinned guinea‐pig smooth muscle. We also investigated which of the three putative substrates at the contractile apparatus ‐ MLCK, caldesmon or r‐MLC ‐ is phosphorylated by PAK1 in smooth muscle tissue. Incubation of Triton‐skinned carotid artery and taenia coli from guinea‐pig with an active mutant of PAK1 in relaxing solution for 30–60 min resulted in inhibition of submaximal force by about 50 %. The mechanism of inhibition of force was studied in the Triton‐skinned taenia coli. In this preparation, inhibition of force was associated with a respective inhibition of r‐MLC phosphorylation. In the presence of the myosin phosphatase inhibitor, microcystin‐LR (10 μm), the rate of contraction and r‐MLC phosphorylation elicited at pCa 6.79 were both decreased. Because under these conditions the rate of r‐MLC phosphorylation is solely dependent on MLCK activity, this result suggests that the inhibitory effect of PAK1 on steady‐state force and r‐MLC phosphorylation is due to inhibition of MLCK. In line with this, we found that MLCK was significantly phosphorylated by PAK1 while there was very little 32P incorporation into caldesmon. PAK1 phosphorylated isolated r‐MLC but not those in the skinned fibres or in purified smooth muscle myosin II. In conclusion, these results suggest that PAK1 attenuates contraction of skinned smooth muscle by phosphorylating and inhibiting MLCK.

Expression of members of the S100 Ca2+-binding protein family in guinea-pig smooth muscle.

The calcium-binding proteins of the S100 family show tissue-specific expression. In this study, the mRNA and protein expression of five S100 calcium-binding proteins was investigated in different guinea-pig smooth muscle preparations. Transcripts of cDNA of S100A1, S100A4, S100A6 and S100A10 were amplified from smooth muscle RNA of neonatal and adult urinary bladder, ileum, aorta and from freshly isolated and cultured urinary bladder smooth muscle cells. S100B was not detectable in smooth muscle RNA, but was seen in control RNA isolated from brain. The pattern of S100 mRNA expression did not change during postnatal development and cell culture of smooth muscle. The structural homology of guinea-pig S100 proteins reverse transcription-polymerase chain reaction (RT-PCR) sequences compared to other species was 85-93% (human), 83-88% (rat) and 81-87% (mouse). Protein expression of S100A4, S100A6 and S100A10 was investigated in aorta, ileum, bladder and cultured bladder smooth muscle cells by Western blot analysis using polyclonal antibodies against guinea-pig-specific S100 immunogenic peptide sequences raised in rabbits. The results show that the proteins S100A4, S100A6 and S100A10 are expressed in the smooth muscle of ileum, bladder and aorta. S100A4 and S100A6 proteins are also expressed in cultured smooth muscle cells. The results of this study suggest that the calcium-binding proteins S100A1, S100A4, S100A6 and S100A10, but not S100B, are expressed in guinea-pig smooth muscle, and could be potentially involved in the regulation of cytoplasmic Ca(2+)-concentration and/or in signal transduction in smooth muscle.

Organization of F-Actin by Fesselin (avian smooth muscle synaptopodin 2)

Fesselin or avian synaptopodin 2 is a member of the synaptopodin family of actin binding proteins. Fesselin promotes G-actin polymerization and the formation of large actin complexes that can be collected by low-speed centrifugation. Because of the potential role of fesselin in some cancers and its effects on actin, we further investigated the effect of fesselin on actin. Fesselin initiated actin polymerization under a variety of conditions, including the virtual absence of salt. Actin filaments formed at low salt concentrations in the presence of fesselin were similar to filaments polymerized in the presence of 100 mM KCl. In both cases, the filaments were long and straight with a common orientation. Highly ordered actin bundles formed with increasing times of incubation. Blockers of actin growth at the barbed end (cytochalasin D and CapZ) did not prevent fesselin from polymerizing actin. Low concentrations of fesselin increased the critical concentration of actin. Both observations are consistent with preferential growth at the pointed end of actin filaments. These results indicate a role of fesselin in organizing cellular actin. These and other results indicate that fesselin is part of a cellular actin organizing center.

Kinase-related protein/telokin inhibits Ca2+-independent contraction in Triton-skinned guinea pig taenia coli

KRP (kinase-related protein), also known as telokin, has been proposed to inhibit smooth muscle contractility by inhibiting the phosphorylation of the rMLC (regulatory myosin light chain) by the Ca2+-activated MLCK (myosin light chain kinase). Using the phosphatase inhibitor microcystin, we show in the present study that KRP also inhibits Ca2+-independent rMLC phosphorylation and smooth muscle contraction mediated by novel Ca2+-independent rMLC kinases. Incubating KRP-depleted Triton-skinned taenia coli with microcystin at pCa>8 induced a slow contraction reaching 90% of maximal force (Fmax) at pCa 4.5 after approximately 25 min. Loading the fibres with KRP significantly slowed down the force development, i.e. the time to reach 50% of Fmax was increased from 8 min to 35 min. KRP similarly inhibited rMLC phosphorylation of HMM (heavy meromyosin) in vitro by MLCK or by the constitutively active MLCK fragment (61K-MLCK) lacking the myosin-docking KRP domain. A C-terminally truncated KRP defective in myosin binding inhibited neither force nor HMM phosphorylation. Phosphorylated KRP inhibited the rMLC phosphorylation of HMM in vitro and Ca2+-insensitive contractions in fibres similar to unphosphorylated KRP, whereby the phosphorylation state of KRP was not altered in the fibres. We conclude that (i) KRP inhibits not only MLCK-induced contractions, but also those elicited by Ca2+-independent rMLC kinases; (ii) phosphorylation of KRP does not modulate this effect; (iii) binding of KRP to myosin is essential for this inhibition; and (iv) KRP inhibition of rMLC phosphorylation is most probably due to the shielding of the phosphorylation site on the rMLC.

Synaptopodin family of natively unfolded, actin binding proteins: physical properties and potential biological functions

The synaptopodin family of proteins consists of at least 3 members: synaptopodin, the synaptopodin 2 proteins, and the synaptopodin 2-like proteins. Each family member has at least 3 isoforms that are produced by alternative splicing. Synaptopodin family members are basic proteins that are rich in proline and have little regular 2° or 3° structure at physiological temperature, pH and ionic strength. Like other natively unfolded proteins, synaptopodin family members have multiple binding partners including actin and other actin-binding proteins. Several members of the synaptopodin family have been shown to stimulate actin polymerization and to bundle actin filaments either on their own or in collaboration with other proteins. Synaptopodin 2 has been shown to accelerate nucleation of actin filament formation and to induce actin bundling. The actin polymerization activity is inhibited by Ca2+-calmodulin. Synaptopodin 2 proteins are localized in Z-bands of striated and heart muscle and dense bodies of smooth muscle cells. Depending on the developmental status and stress, at least one member of the synaptopodin family can occupy nuclei of some cells. Members of the synaptopodin 2 subfamily have been implicated in cancers.

Acrylodan-Labeled Smooth Muscle Tropomyosin Reports Differences in the Effects of Troponin and Caldesmon in the Transition from the Active State to the Inactive State

Changes in the orientation of tropomyosin on actin are important for the regulation of striated muscle contraction and could also be important for smooth muscle regulation. We showed earlier that acrylodan-labeled skeletal muscle tropomyosin reports the kinetics of the reversible transitions among the active, intermediate, and inactive states when S1 is rapidly detached from actin-tropomyosin. We now show that acrylodan-labeled smooth muscle tropomyosin reports similar transitions among states of actin-tropomyosin. When S1 was rapidly detached from actin-smooth muscle tropomyosin, there was a rapid decrease in acrylodan-tropomyosin fluorescence as the intermediate state became populated. The rate constant for this process was >600 s(-1) at temperatures near 5 °C. In the presence of skeletal troponin and EGTA, the decrease in fluorescence was followed by the redevelopment of fluorescence as the inactive state became populated. The apparent rate constant for the fluorescence increase was 14 s(-1) at 5 °C. Substituting smooth muscle caldesmon for skeletal muscle troponin produced a similar decrease and re-increase in fluorescence, but the apparent rate constant for the increase was >10 times that observed with troponin. Furthermore, the fluorescence increase was correlated with an increase in the extent of caldesmon attachment as S1-ATP dissociated. Although the measured rate constant appeared to reflect the rate-limiting transition for inactivation, it is unclear if the fluorescence change resulted from caldesmon binding, the movement of tropomyosin over actin, or both.

Aging-related alterations in eNOS and nNOS responsiveness and smooth muscle reactivity of murine basilar arteries are modulated by apocynin and phosphorylation of myosin phosphatase targeting subunit-1.

Aging causes major alterations of all components of the neurovascular unit and compromises brain blood supply. Here, we tested how aging affects vascular reactivity in basilar arteries from young (<10 weeks; y-BA), old (>22 months; o-BA) and old (>22 months) heterozygous MYPT1-T-696A/+ knock-in mice. In isometrically mounted o-BA, media thickness was increased by ∼10% while the passive length tension relations were not altered. Endothelial denudation or pan-NOS inhibition (100 µmol/L L-NAME) increased the basal tone by 11% in y-BA and 23% in o-BA, while inhibition of nNOS (1 µmol/L L-NPA) induced ∼10% increase in both ages. eNOS expression was ∼2-fold higher in o-BA. In o-BA, U46619-induced force was augmented (pEC50 ∼6.9 vs. pEC50 ∼6.5) while responsiveness to DEA-NONOate, electrical field stimulation or nicotine was decreased. Basal phosphorylation of MLC20-S19 and MYPT1-T-853 was higher in o-BA and was reversed by apocynin. Furthermore, permeabilized o-BA showed enhanced Ca2+-sensitivity. Old T-696A/+ BA displayed a reduced phosphorylation of MYPT1-T696 and MLC20, a lower basal tone in response to L-NAME and a reduced eNOS expression. The results indicate that the vascular hypercontractility found in o-BA is mediated by inhibition of MLCP and is partially compensated by an upregulation of endothelial NO release.

Neonatal mouse ileum: functional properties and protein composition of the contractile machinery

Background:Immature motility of the ileum may contribute to life-threatening diseases. Little is known about the normal biomechanics of the neonatal ileum in relation to the protein composition of its contractile machinery.Methods:We analyzed the tissue architecture, the biomechanics in intact and β-escin-permeabilized preparations, and the protein composition in neonatal (P0) and adult murine ileum.Results:Muscle thickness of the P0 ileum was −50% of the adult ileum and passive compliance was higher. Carbachol- and KCl-elicited contractions were tonic rather than phasic as in the adult. Ca2+ sensitivity was higher and relaxation rate was slower in β-escin-permeabilized P0 compared with adult ileum. The expression level of β-actin relative to α-actin was higher, and those of total actin, myosin, myosin light chain kinase, the catalytic subunit of myosin phosphatase and telokin were lower compared with the adult. The expression level of MYPT1 was similar, but P0 ileum expressed only the M133; the adult ileum also expressed the M130 isoform.Conclusion:The mechanical features and protein composition of the P0 ileum are similar to those of adult tonic smooth muscles. We propose that this is highly adaptive during fetal life allowing the small intestine to act predominantly as a container.Pediatric Research (2014); 76 3, 252–260. doi:10.1038/pr.2014.91

The involvement of phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and MYPT1 isoform expression in NO/cGMP mediated differential vasoregulation of cerebral arteries compared to systemic arteries

Constitutive release of NO blunts intrinsic and stimulated contractile activity in cerebral arteries (CA). Here, we explored whether phosphorylation and expression levels of the PKG‐sensitive, leucine zipper positive (LZ+) splice variants of the regulatory subunit of myosin phosphatase (MYPT1) are involved and whether its expression is associated with higher cGMP sensitivity.