Epithelial myosin light chain kinase expression and activity are upregulated in inflammatory bowel disease

The intestinal epithelial barrier is frequently disrupted in inflammatory bowel disease (IBD) and this has been proposed to play a role in disease pathogenesis and reactivation. In vitro studies show that cytokine-induced epithelial barrier dysfunction can be mediated by increased myosin light chain kinase (MLCK) expression and subsequent myosin II regulatory light chain (MLC) phosphorylation. However, this has never been examined in human disease. The aim of these studies, therefore, was to determine whether MLCK is upregulated in the intestinal epithelium of IBD patients. MLCK expression and MLC phosphorylation in human intestinal resection and biopsy specimens were determined by quantitative immunofluorescence microscopy and correlated with clinical and histopathological data. The data show that ileal epithelial MLCK expression was mildly upregulated in inactive IBD. Expression increased further in active disease, with progressive increases in MLCK expression correlating positively with histological disease activity. This correlation between activity and MLCK expression was also seen in individual patients where areas of differing disease activity were analyzed. Colonic epithelial MLCK expression was similarly increased in active IBD and these increases also correlated positively with disease activity, both in individual patients and the overall study group. To evaluate MLCK enzymatic activity, MLC phosphorylation was assessed in snap-frozen colon biopsies. MLC phosphorylation was significantly increased in biopsies with active, but not inactive, IBD. Therefore, these data show that MLCK expression and enzymatic activity are increased in IBD. Moreover, the correlation with disease activity suggests that MLCK upregulation may contribute to barrier dysfunction and IBD pathogenesis.     

Novel polymorphisms in the myosin light chain kinase gene confer risk for acute lung injury

The genetic basis of acute lung injury (ALI) is poorly understood. The myosin light chain kinase (MYLK) gene encodes the nonmuscle myosin light chain kinase isoform, a multifunctional protein involved in the inflammatory response (apoptosis, vascular permeability, leukocyte diapedesis). To examine MYLK as a novel candidate gene in sepsis-associated ALI, we sequenced exons, exon-intron boundaries, and 2 kb of 5' UTR of the MYLK, which revealed 51 single-nucleotide polymorphisms (SNPs). Potential association of 28 MYLK SNPs with sepsis-associated ALI were evaluated in a case-control sample of 288 European American subjects (EAs) with sepsis alone, subjects with sepsis-associated ALI, or healthy control subjects, and a sample population of 158 African American subjects (AAs) with sepsis and ALI. Significant single locus associations in EAs were observed between four MYLK SNPs and the sepsis phenotype (P<0.001), with an additional SNP associated with the ALI phenotype (P=0.03). A significant association of a single SNP (identical to the SNP identified in EAs) was observed in AAs with sepsis (P=0.002) and with ALI (P=0.01). Three sepsis risk-conferring haplotypes in EAs were defined downstream of start codon of smooth muscle MYLK isoform, a region containing putative regulatory elements (P<0.001). In contrast, multiple haplotypic analyses revealed an ALI-specific, risk-conferring haplotype at 5' of the MYLK gene in both European and African Americans and an additional 3' region haplotype only in African Americans. These data strongly implicate MYLK genetic variants to confer increased risk of sepsis and sepsis-associated ALI.     

Hindlimb suspension induces the expression of multiple myosin heavy chain isoforms in single fibres of the rat soleus muscle

The aim of our experiment was to test the hypothesis that the performance of maximal isometric exercise every 20 s would reduce the intermediate frequency force, i.e. the force that appears while stimulating the muscle at 15 and 20 Hz, and would produce less decrease the force at 10 and 50 Hz, while Pt would increase. Such changes in stimulated force should demonstrate the coexistence of potentiation, low frequency fatigue (LFF) and `post-contractile depression' (PCD). The quadriceps muscle of 14 healthy men (aged 19–37 years) was studied. The results have shown, that during isometric exercise of maximal intensity there was significant (P < 0.05) decrease in P15 and P20, increase in Pt, however, MVC and P10 and P50 was unchanged (P > 0.05). LFF manifested itself most significantly which is evident from decrease in P20/P50. During recovery after work there was significant increase in LFF and decrease in P50 which is indicative of the manifestation of PCD. Besides, there was significant (P < 0.05) decrease immediately after exercise in RTP20 and RTP50, while no changes in T50 and RT. There were no significant changes (P > 0.05) however, either in RTP20 and RTP50 or in T50 and RT 20 min after exercise if compared to the initial and immediately post-exercise values.     

Polymorphisms in the myosin light chain kinase gene that confer risk of severe sepsis are associated with a lower risk of asthma

BACKGROUND: Myosin light chain kinase (MYLK) is a multifunctional protein involved in regulation of airway hyperreactivity and other activities relevant to asthma. OBJECTIVE: To determine the role of MYLK gene variants in asthma among African Caribbean and African American populations. METHODS: We performed association tests between single nucleotide polymorphisms (SNPs) in the MYLK gene and asthma susceptibility and total serum IgE concentrations in 2 independent, family-based populations of African descent. Previously we identified variants/haplotypes in MYLK that confer risk for sepsis and acute lung injury; we compared findings from our asthma populations to findings in the African American sepsis and acute lung injury groups. RESULTS: Significant associations between MYLK SNPs and asthma and total serum IgE concentrations were observed in the African Caribbean families: a promoter SNP (rs936170) in the smooth muscle form gave the strongest association (P = .009). A haplotype including rs936170 corresponding to the actin-binding activity of the nonmuscle and smooth muscle forms was negatively associated with asthma (eg, decreased risk) in both the American (P = .005) and Caribbean families (P = .004), and was the same haplotype that conferred risk for severe sepsis (P = .002). RNA expression studies on PBMCs and rs936170 suggested a significant decrease in MYLK expression among patients with asthma with this variant (P = .025). CONCLUSION: MYLK polymorphisms may function as a common genetic factor in clinically distinct diseases involving bronchial smooth muscle contraction and inflammation. CLINICAL IMPLICATIONS: Genetic variants in MYLK are significantly associated with both asthma and sepsis in populations of African ancestry.     

The effect of skeletal myosin light chain kinase gene ablation on the fatigability of mouse fast muscle

Contraction-induced activation of a skeletal muscle specific Ca(2+) and calmodulin dependent myosin light chain kinase (skMLCK) catalyzes phosphorylation of the myosin regulatory light chain (RLC), a reaction that potentiates twitch force. The purpose of this study was to test the effect of skMLCK gene ablation on the fatigability of mouse extensor digitorum longus (EDL) muscle (in vitro at 25°C). Muscles were isolated from wildtype (WT, n = 10-12) and skeletal MLCK knockout (skMLCK KO, n = 10-12) mice and fatigued using a protocol consisting of 5 min of repeated tetanic stimulation (150 Hz for 1000 ms every 5 s). Both twitch (P(t)) and tetanic (P(o)) force as well as unloaded shortening velocity (V(o)) were assessed before, during and after fatiguing stimulation. Fatiguing stimulation increased RLC phosphorylation in WT but not skMLCK KO muscles (16 ± 0.01-0.63 ± 0.02 and 0.07 ± 0.02-0.08 ± 0.02 mol phos mol RLC, respectively). Although P(t) was potentiated above baseline in both WT and KO muscles, this increase was greater in WT than in KO muscles (to 1.37 ± 0.05 vs. 1.14 ± 0.02 of unpotentiated values, respectively). The difference in P(t) persisted until P(o) had been diminished to ~60% of baseline and thereafter P(t) declined to similar levels in both WT and KO muscles (to ~35% of initial). Overall, the time-course and decline in P(o) for WT and KO was similar (reduced to 0.20 ± 0.01 and 0.20 ± 0.01 of baseline, respectively) (P < 0.05). Initial values for V(o) were similar between WT and KO muscles and, moreover, the fatigue related decline in Vo was similar for both muscle genotypes (P < 0.05). Thus, our results demonstrate that skMLCK--catalyzed RLC phosphorylation augments isometric twitch force during moderate, but not severe, levels of fatigue.