Ulf Malmqvist

Correlation between isoform composition of the 17 kDa myosin light chain and maximal shortening velocity in smooth muscle

The relation between the isoform distribution of the myosin 17 kDa essential light chain (LC17) and the mechanical properties of smooth muscle was investigated. The relative content of the basic (LC17b) and acidic (LC17a) isoelectric variants of the 17 kDa myosin light chain was determined in different mammalian smooth muscle tissues. The relative content of LC17b varied between muscles: rabbit rectococcygeus 0%, rabbit trachea 5%, guinea-pig taenia coli 21%, rat uterus 38%, rabbit aorta 56% and rat aorta 60%. The rate of tension development was determined following photolysis of cagedadenosine triphosphate (ATP) in skinned fibres activated with thiophosphorylation of the regulatory light chains. The half-time for force development was 0.67 s in rabbit rectococcygeus, 1.6 s in rabbit trachea, 1.13 s in guineapig taenia coli and 1.38 s in rabbit aorta. The maximal shortening velocity (vmax) was determined with the isotonic quick release technique in skinned fibre preparations activated with thiophosphorylation. vmax was 0.25 muscle lengths per second (ML/s) in rabbit rectococcygeus, 0.24 ML/s in rabbit trachea, 0.17 ML/s in guinea-pig taenia coli, 0.11 ML/s in rat uterus and 0.03 ML/s in rabbit aorta. The range of variation in vmax between muscles was larger than in the half-time for force development. The inverse relationship between vmax and the relative content of LC17b in the investigated muscles suggests that the type of essential myosin light chain influences the vmax in smooth muscle.

Novel subgroups of adult-onset diabetes and their association with outcomes: a data-driven cluster analysis of six variables

BACKGROUND Diabetes is presently classified into two main forms, type 1 and type 2 diabetes, but type 2 diabetes in particular is highly heterogeneous. A refined classification could provide a powerful tool to individualise treatment regimens and identify individuals with increased risk of complications at diagnosis. METHODS We did data-driven cluster analysis (k-means and hierarchical clustering) in patients with newly diagnosed diabetes (n=8980) from the Swedish All New Diabetics in Scania cohort. Clusters were based on six variables (glutamate decarboxylase antibodies, age at diagnosis, BMI, HbA1c, and homoeostatic model assessment 2 estimates of β-cell function and insulin resistance), and were related to prospective data from patient records on development of complications and prescription of medication. Replication was done in three independent cohorts: the Scania Diabetes Registry (n=1466), All New Diabetics in Uppsala (n=844), and Diabetes Registry Vaasa (n=3485). Cox regression and logistic regression were used to compare time to medication, time to reaching the treatment goal, and risk of diabetic complications and genetic associations. FINDINGS We identified five replicable clusters of patients with diabetes, which had significantly different patient characteristics and risk of diabetic complications. In particular, individuals in cluster 3 (most resistant to insulin) had significantly higher risk of diabetic kidney disease than individuals in clusters 4 and 5, but had been prescribed similar diabetes treatment. Cluster 2 (insulin deficient) had the highest risk of retinopathy. In support of the clustering, genetic associations in the clusters differed from those seen in traditional type 2 diabetes. INTERPRETATION We stratified patients into five subgroups with differing disease progression and risk of diabetic complications. This new substratification might eventually help to tailor and target early treatment to patients who would benefit most, thereby representing a first step towards precision medicine in diabetes. FUNDING Swedish Research Council, European Research Council, Vinnova, Academy of Finland, Novo Nordisk Foundation, Scania University Hospital, Sigrid Juselius Foundation, Innovative Medicines Initiative 2 Joint Undertaking, Vasa Hospital district, Jakobstadsnejden Heart Foundation, Folkhälsan Research Foundation, Ollqvist Foundation, and Swedish Foundation for Strategic Research.

Stretch-Dependent Modulation of Contractility and Growth in Smooth Muscle of Rat Portal Vein

Increased intraluminal pressure of the rat portal vein in vivo causes hypertrophy and altered contractility in 1 to 7 days. We have used organ cultures to investigate mechanisms involved in this adaptation to mechanical load. Strips of rat portal vein were cultured for 3 days, either undistended or loaded by a weight. Length-force relations were shifted toward longer length in stretched cultured veins compared with freshly dissected veins, whereas the length-force relations of unstretched cultured veins were shifted in the opposite direction. This occurred after culture either with or without 10% FCS to promote growth. The wet weight of loaded veins increased by 56% in the presence of FCS, whereas that of undistended control veins increased by 24%. No weight increase was seen in serum-free culture. The dry/wet weight ratio decreased during culture with FCS but was not affected by stretch. Electron microscopy revealed increased cell cross-sectional area in stretched relative to unstretched veins, and protein contents were greater, as were [(3)H]thymidine and [(3)H]leucine incorporation rates. Growth responses were associated with the activation of stretch-sensitive extracellular signal-regulated kinases 1 and 2 and were inhibited by herbimycin A and PD 98059, inhibitors of extracellular signal-regulated kinases 1 and 2. The results demonstrate that by culture of whole vascular tissue, smooth muscle cells are maintained in the contractile phenotype and respond to stretch with a physiological adaptation involving hypertrophy/hyperplasia and remodeling of the contractile system, similar to that in vivo. Mechanical stimulation and growth factors are both required for functionally significant growth.

Isoform distribution and tissue contents of contractile and cytoskeletal proteins in hypertrophied smooth muscle from rat portal vein.

Growth of the smooth muscle in the rat portal vein was initiated by an increased transmural pressure. After 7 days, the cross-sectional area of the vessel wall and the maximal active force of the longitudinal muscle layer had increased twofold. Electron microscopy showed that the cell cross-sectional area was increased, suggesting cellular hypertrophy. Increased amounts of intermediate (10 nm) filaments were observed in the hypertrophied cells. The hypertrophied vessels had decreased DNA content per unit wet weight compared with the control vessels (hypertrophied, 1.5 +/- 0.1; control, 1.9 +/- 0.1 micrograms/mg; p less than 0.01). Protein composition was studied with electrophoretic methods. Compared with control preparations the hypertrophied veins had similar myosin and actin contents per unit wet weight (myosin: hypertrophied, 4.4 +/- 0.8; control, 5.9 +/- 0.9; actin: hypertrophied 12.2 +/- 0.6; control, 11.8 +/- 1.0 mg/g). Two different forms of the myosin heavy chain were detected with 5% sodium dodecyl sulfate-polyacrylamide gels. The proportion of the lower molecular weight heavy chain relative to total heavy chain content was about 30% and similar in both preparations. The relation filamin/myosin was increased in the hypertrophied vessels. Pyrophosphate gel electrophoresis revealed two protein bands, with an increase in the slower migrating band in the hypertrophied vessels possibly reflecting an increase in filamin content in the extracts. In the control portal vein alpha-actin is the dominating isoform constituting about 55% of total actin. In hypertrophied vessels, alpha-actin decreased (by 15%) and gamma-actin increased (by 20%). The portal vein contained desmin and vimentin in a ratio of about 6:1. The hypertrophied vessels showed a marked increase in the amount of these proteins (desmin/actin: hypertrophied, 0.32; control, 0.14). In conclusion, during pressure-induced growth of the portal vein, contractile protein contents increase in proportion to the increase in weight. A change in isoforms of actin occurs but no evidence for a change in myosin isoforms was found. The structural proteins increase relative to tissue weight, possibly associated with the increased number of intermediate filaments demonstrated with electron microscopy.

Long-term effects of Ca(2+) on structure and contractility of vascular smooth muscle

Culture of dispersed smooth muscle cells is known to cause rapid modulation from the contractile to the synthetic cellular phenotype. However, organ culture of smooth muscle tissue, with maintained extracellular matrix and cell-cell contacts, may facilitate maintenance of the contractile phenotype. To test the influence of culture conditions, structural, functional, and biochemical properties of rat tail arterial rings were investigated after culture. Rings were cultured for 4 days in the absence and presence of 10% FCS and then mounted for physiological experiments. Intracellular Ca2+concentration ([Ca2+]i) after stimulation with norepinephrine was similar in rings cultured with and without FCS, whereas force development after FCS was decreased by >50%. The difference persisted after permeabilization with β-escin. These effects were associated with the presence of vasoconstrictors in FCS and were dissociated from its growth-stimulatory action. FCS treatment increased lactate production but did not affect ATP, ADP, or AMP contents. The contents of actin and myosin were decreased by culture but similar for all culture conditions. There was no effect of FCS on calponin contents or myosin SM1/SM2 isoform composition, nor was there any appearance of nonmuscle myosin. FCS-stimulated rings showed evidence of cell degeneration not found after culture without FCS or with FCS + verapamil (1 μM) to lower [Ca2+]i. The decreased force-generating ability after culture with FCS is thus associated with increased [Ca2+]iduring culture and not primarily caused by growth-associated modulation of cells from the contractile to the synthetic phenotype.

Determination of Nicotine Absorption from Multiple Tobacco Products and Nicotine Gum

Introduction: Snus is a smokeless tobacco product traditionally used in Scandinavia and available in pouched or loose forms. The objective of this study was to determine nicotine absorption for current pouched and loose snus products in comparison with a cigarette and an over-the-counter nicotine gum. Methods: We conducted an open-label, randomized, 6-way, crossover study involving 20 healthy snus and cigarette users. One of 6 products (2 pouched snus, 2 weights of loose snus, a cigarette, and a nicotine gum) was administered at each of 6 visits. Blood samples were taken at intervals over 120 min and sensory perception assessed by questionnaire. Results: For the 4 smokeless tobacco products and the nicotine gum, blood plasma levels of nicotine were ranked according to total nicotine content as follows: loose snus (27.1 mg nicotine) > pouched snus (14.7 mg nicotine) > loose snus (10.8 mg nicotine) = pouched snus (10.7 mg nicotine) > nicotine gum (4.2 mg nicotine). The area under the plasma concentration–time curve (AUC) and maximum plasma concentration (Cmax) of nicotine ranged from 26.9 to 13.1 ng.h/ml and 17.9 to 9.1 ng.h/ml, respectively across all the products. Nicotine was absorbed more rapidly from the cigarette but systemic exposure was within the range of the smokeless tobacco products (AUC = 14.8 ng.h/ml; Cmax = 12.8 ng.h/ml). Conclusions: This study has generated new information on comparative nicotine absorption from a cigarette, loose snus, and pouched snus typical of products sold in Scandinavia. The similar nicotine absorption for 1 g portions of loose and pouched snus with approximately 11 mg of nicotine indicate that absorption kinetics were dependent on quantity of tobacco by weight and total nicotine content rather than product form.

Cytoskeletal and Contractile Proteins in Detrusor Smooth Muscle from Bladders with Outlet Obstruction—a Comparative Study in RAT and MAN

Detrusor biopsies were obtained from patients with urinary outlet obstruction due to prostatic enlargement and from age-matched control patients. The relative amounts of actin and myosin and their isoforms, as well as desmin and filamin were determined and compared with corresponding results from bladders from control rats and rats with 10 days of experimental outlet obstruction of the urinary bladder. In the human control detrusor the actin/myosin ratio was similar to that in the control rat. The isoform distribution of the myosin heavy chains differed between man and rat. In the biopsies from the patients with outlet obstruction and in the obstructed rat bladders the actin/myosin ratio was increased. A change in the myosin heavy chain distribution in the obstructed bladders was observed for both species. The filamin/actin ratio increased significantly in the obstructed rat bladders and tended to increase in the obstructed human bladders. Desmin was the dominating intermediate filament protein. The desmin/actin ratio increased in obstructed bladders in man and in rat.

Effects of 2,3-butanedione monoxime on activation of contraction and crossbridge kinetics in intact and chemically skinned smooth muscle fibres from guinea pig taenia coli

SummaryThe effects of 2,3-butanedione monoxime (BDM) were studied in smooth muscle fibres from guinea pig taenia coli. In intact muscle, active force during contractions induced by high-K+ was inhibited by about 10% in 1 mM BDM and by approximately 70% in 10 mM BDM. Intracellular [Ca2+] during contraction, measured with the fura-2 technique, was reduced in the presence of BDM. The reduction in force and [Ca2+] in the presence of 1 and 10 mM BDM could be reproduced by reduction in extracellular Ca2+, suggesting that BDM influences the Ca2+ entry or release. In skinned muscle preparations, BDM decreased the Ca2+ sensitivity of active force. This change could be explained by a decreased level of myosin light chain phosphorylation. In fibres maximally activated by thiophosphorylation, the effect of BDM on force occurred at higher concentrations; 10 mM gave no reduction of force and 60 mM 15% reduction. The maximal shortening velocity (Vmax) and force were unaffected by 30 mM BDM in thiophosphorylated muscle and decreased almost in parallel in Ca2+-activated contractions. The present results suggest that BDM inhibits myosin light chain phosphorylation, directly decreases force generation at the crossbridge level and inhibits the Ca2+ translocation in smooth muscle. The effect on force in skinned fibres is observed at higher BDM concentrations than those reported to be required for inhibition of force in striated muscle. The inhibition of force in intact smooth muscle could be explained by an influence on Ca2+ translocation.

The effects of caldesmon extraction on mechanical properties of skinned smooth muscle fibre preparations

The role of caldesmon in the regulation of smooth muscle contraction was investigated in chemically skinned smooth muscle fibres from the guineapig taenia coli. A 19-kDa C-terminal fragment of caldesmon gave a minor (<5%) reduction of force in fully thiophosphorylated fibres, but reduced force by about 50% at intermediate activation levels without affecting the level of light chain phosphorylation. An extraction procedure was developed using incubation in solutions containing high Mg2+ concentrations. Protein analysis revealed a selective decrease in the amount of caldesmon in the fibres. Maximal active force per cross-sectional area was unaffected. The Ca2+ dependence of active force was shifted towards lower Ca2+ concentrations and became less steep. The effects of extraction of caldesmon could in part be reversed by incubation in a solution containing purified caldesmon. The results are consistent with the hypothesis that caldesmon in smooth muscle thin filaments inhibits force generation and plays a role in regulating cooperative attachment of cross-bridges at sub-maximal levels of activation in smooth muscle.

Lactate dehydrogenase activity and isoform distribution in normal and hypertrophic smooth muscle tissue from the rat

The lactate dehydrogenase (LDH) activity and isoform distribution of LDH were investigated in tissue samples from the rat portal vein, aorta and urinary bladder. In addition, samples were obtained from hypertrophic urinary bladder. The total LDH activity per unit smooth muscle volume was higher in the urinary bladder compared to that in portal vein and aorta. Five LDH isoforms, reflecting different combinations of the two polypeptide chains denoted H and M, could be separated by agarose gel electrophoresis. The aorta contained more of the H form compared to the portal vein and urinary bladder. This difference suggests that the aorta, which is a slow smooth muscle, is more adapted for aerobic metabolism than the faster muscles of portal vein and urinary bladder. In the hypertrophic urinary bladder a shift in LDH isoform pattern towards less of the H form was found, which correlates with a better maintenance of contraction in anoxia in this type of hypertrophic smooth muscle.