Action and therapeutic targets of myosin light chain kinase, an important cardiovascular signaling mechanism.

The global incidence of cardiac diseases is increasing, imposing a substantial socioeconomic burden on healthcare systems. The pathogenesis of cardiovascular disease is complex and not fully understood, and the physiological function of the heart is inextricably linked to well-regulated cardiac muscle movement. Myosin light chain kinase (MLCK) is essential for myocardial contraction and diastole, cardiac electrophysiological homeostasis, vasoconstriction of vascular nerves and blood pressure regulation. In this sense, MLCK appears to be an attractive therapeutic target for cardiac diseases. MLCK participates in myocardial cell movement and migration through diverse pathways, including regulation of calcium homeostasis, activation of myosin light chain phosphorylation, and stimulation of vascular smooth muscle cell contraction or relaxation. Recently, phosphorylation of myosin light chains has been shown to be closely associated with the activation of myocardial exercise signaling, and MLCK mediates systolic and diastolic functions of the heart through the interaction of myosin thick filaments and actin thin filaments. It works by upholding the integrity of the cytoskeleton, modifying the conformation of the myosin head, and modulating innervation. MLCK governs vasoconstriction and diastolic function and is associated with the activation of adrenergic and sympathetic nervous systems, extracellular transport, endothelial permeability, and the regulation of nitric oxide and angiotensin II. Additionally, MLCK plays a crucial role in the process of cardiac aging. Multiple natural products/phytochemicals and chemical compounds, such as quercetin, cyclosporin, and ML-7 hydrochloride, have been shown to regulate cardiomyocyte MLCK. The MLCK-modifying capacity of these compounds should be considered in designing novel therapeutic agents. This review summarizes the mechanism of action of MLCK in the cardiovascular system and the therapeutic potential of reported chemical compounds in cardiac diseases by modifying MLCK processes.

Inhibition of non-muscular myosin light chain kinase accelerates the clearance of inflammatory cells by promoting the lysosome-mediated cell death.

Infections like COVID-19 are the primary cause of death around the world because they can cause acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and sepsis. Inflammatory cells serve as crucial protective barriers in these diseases. However, excessive accumulation of inflammatory cells is also one of the major causes of organ damage. The non-muscular myosin light chain kinase (nmMLCK) plays crucial of cytoskeletal components involved in endothelial cell-matrix and cell-cell adhesion, integrity, and permeability. Our previous investigations found that ML-7, a specific inhibitor of MLCK, promoted neutrophil apoptosis through various signaling pathways. In this study, we found that knockout of MLCK significantly promote apoptosis of neutrophils and macrophages in the BALF of the LPS-induced ALI, meanwhile it had no effect on the apoptosis of neutrophils in the circulatory system. RNA-sequencing revealed that the effect of MLCK knockout in inducing apoptosis of inflammatory cells was mediated through lysosomes. Administering ML-7 into the lungs significantly promoted neutrophil apoptosis, accelerating their clearance. In the LPS- or CLP-induced sepsis models, ML-7 administration significantly improves the apoptosis of inflammatory cells, especially neutrophils, at the infection site but had no impact on neutrophils in the circulatory system. ML-7 also significantly improved the survival rate of mice with LPS- or CLP-induced sepsis. Taken together, we found that MLCK plays a crucial role in the survival of inflammatory cells at the infection site. Inhibiting MLCK significantly induces apoptosis of inflammatory cells at the infection site, promoting inflammation resolution, with no impact of the circulatory system.

Inhibition of the MLCK/MLC2 pathway protects against intestinal heat stroke-induced injury in rats.

Intestinal barrier dysfunction often exists in the heat stroke (HS) pathological process, which increases intestinal permeability and induces endotoxemia. The upregulation of MLCK is a crucial player affecting intestinal permeability. This study aimed to explore whether inhibiting myosin light chain kinase (MLCK) can improve HS-induced intestinal injury in rats. Twelve-week-old Wistar male rats were divided into three groups: the control group, the HS model group, and the treatment group [HS model + ML-7 (MLCK inhibitor)]. HS impaired the tight junctions in the rat gut and increased permeability. Additionally, increased inflammatory factors in serum, activation of apoptosis, and downregulation of tight junction proteins were observed in intestinal cells. ML-7 significantly inhibited the MLCK/p-MLC2 signaling pathway, increased the expression of tight junction proteins, reduced intestinal permeability, reduced apoptosis and alleviated the intestinal damage caused by HS. ML-7 inhibited HS-induced apoptosis of intestinal epithelial cells by regulating the ERK/p38/HSP70 axis. Furthermore, inhibition of MLCK upregulated HSP70 expression through activation of the ERK pathway and inhibited cell apoptosis by abolishing the p38 MAPK pathway. In conclusion, inhibiting the MLCK/p-MLC2 signaling pathway reduces HS-induced intestinal permeability and protects the intestinal mucosal barrier.

Comparison of Rheological Properties of Healthy versus Dupuytren Fibroblasts When Treated with a Cell Contraction Inhibitor by Atomic Force Microscope.

Mechanical properties of healthy and Dupuytren fibroblasts were investigated by atomic force microscopy (AFM). In addition to standard force curves, rheological properties were assessed using an oscillatory testing methodology, in which the frequency was swept from 1 Hz to 1 kHz, and data were analyzed using the structural damping model. Dupuytren fibroblasts showed larger apparent Young's modulus values than healthy ones, which is in agreement with previous results. Moreover, cell mechanics were compared before and after ML-7 treatment, which is a myosin light chain kinase inhibitor (MLCK) that reduces myosin activity and hence cell contraction. We employed two different concentrations of ML-7 inhibitor and could observe distinct cell reactions. At 1 µM, healthy and scar fibroblasts did not show measurable changes in stiffness, but Dupuytren fibroblasts displayed a softening and recovery after some time. When increasing ML-7 concentration (3 µM), the majority of cells reacted, Dupuytren fibroblasts were the most susceptible, not being able to recover from the drug and dying. These results suggested that ML-7 is a potent inhibitor for MLCK and that myosin II is essential for cytoskeleton stabilization and cell survival.

Tumor Necrosis Factor Alpha Effects on the Porcine Intestinal Epithelial Barrier Include Enhanced Expression of TNF Receptor 1.

Tumor necrosis factor alpha (TNFα) has been shown to impair the intestinal barrier, inducing and maintaining inflammatory states of the intestine. The aim of the current study was to analyze functional, molecular and regulatory effects of TNFα in a newly established non-transformed jejunal enterocyte model, namely IPEC-J2 monolayers. Incubation with 1000 U/mL TNFα induced a marked decrease in transepithelial electrical resistance (TEER), and an increase in permeability for the paracellular flux marker [3H]-D-mannitol compared to controls. Immunoblots revealed a significant decrease in tight junction (TJ) proteins occludin, claudin-1 and claudin-3. Moreover, a dose-dependent increase in the TNF receptor (TNFR)-1 was detected, explaining the exponential nature of pro-inflammatory effects, while TNFR-2 remained unchanged. Recovery experiments revealed reversible effects after the removal of the cytokine, excluding apoptosis as a reason for the observed changes. Furthermore, TNFα signaling could be inhibited by the specific myosin light chain kinase (MLCK) blocker ML-7. Results of confocal laser scanning immunofluorescence microscopy were in accordance with all quantitative changes. This study explains the self-enhancing effects of TNFα mediated by MLCK, leading to a differential regulation of TJ proteins resulting in barrier impairment in the intestinal epithelium.

A Novel Tigecycline Adjuvant ML-7 Reverses the Susceptibility of Tigecycline-Resistant Klebsiella pneumoniae.

The increasing incidence of tigecycline resistance undoubtedly constitutes a serious threat to global public health. The combination therapies had become the indispensable strategy against this threat. Herein, 11 clinical tigecycline-resistant Klebsiella pneumoniae which mainly has mutations in ramR, acrR, or macB were collected for tigecycline adjuvant screening. Interestingly, ML-7 hydrochloride (ML-7) dramatically potentiated tigecycline activity. We further picked up five analogs of ML-7 and evaluated their synergistic activities with tigecycline by using checkerboard assay. The results revealed that ML-7 showed certain synergy with tigecycline, while other analogs exerted attenuated synergistic effects among tigecycline-resistant isolates. Thus, ML-7 was selected for further investigation. The results from growth curves showed that ML-7 combined with tigecycline could completely inhibit the growth of bacteria, and the time-kill analysis revealed that the combination exhibited synergistic bactericidal activities for tigecycline-resistant isolates during 24 h. The ethidium bromide (EtBr) efflux assay demonstrated that ML-7 could inhibit the functions of efflux pump. Besides, ML-7 disrupted the proton motive force (PMF) via increasing ΔpH, which in turn lead to the inhibition of the functions of efflux pump, reduction of intracellular ATP levels, as well as accumulation of ROS. All of which promoted the death of bacteria. And further transcriptomic analysis revealed that genes related to the mechanism of ML-7 mainly enriched in ABC transporters. Taken together, these results revealed the potential of ML-7 as a novel tigecycline adjuvant to circumvent tigecycline-resistant Klebsiella pneumoniae.

Myosin light chain kinase inhibitor ML7 improves vascular endothelial dysfunction and permeability via the mitogen-activated protein kinase pathway in a rabbit model of atherosclerosis.

Endothelial dysfunction (ED) and hyperpermeability are considered as the initiating steps in early atherosclerosis. Phosphorylation of myosin light chain (MLC) is key to cause vascular hyperpermeability via endothelial cell contraction. However, it is unclear whether MLC phosphorylation can also regulate the balance between contraction and relaxation of endothelial cells, thereby affecting endothelium-dependent diastolic function and leading to ED. The present study investigated relationships between ED and MLC phosphorylation and underlying mechanisms. Twenty-four male New Zealand white rabbits were randomly divided into three groups: control, AS, and ML7 (MLCK inhibitor) groups, and fed with normal diet, high-fat diet (HFD), and HFD plus oral ML7 (1 mg/kg daily) respectively. HFD-fed rabbits showed typical atheromatous lesions and endothelial hyperpermeability, and these lesions could be partly reversed following ML7 therapy. Western blotting revealed significant increased expression of myosin light chain kinase (MLCK) and phosphorylation of MLC, JNK, and ERK in the arterial wall of rabbits in the AS group compared with those of the control group (p < 0.05), whereas the ML7 group showed markedly decreased levels of these proteins compared with the AS group (p < 0.05). The endothelium-dependent relaxation rate was significantly reduced both in vitro and in vivo in AS group, and was improved using ML7 therapy. Taken together, these results indicate that MLCK expression and subsequent MLC phosphorylation increase vascular endothelial permeability and endothelium-dependent diastolic dysfunction by promoting endothelial cell contraction, which may be initiated by the activation of the MAP/ERK (MEK) and MAP/JNK (MEK) pathways.

The Contractile Apparatus Is Essential for the Integrity of the Blood-Brain Barrier After Experimental Subarachnoid Hemorrhage.

Development of vasogenic brain edema is a key event contributing to mortality after subarachnoid hemorrhage (SAH). The precise underlying mechanisms at the neurovascular level that lead to disruption of the blood-brain barrier (BBB) are still unknown. Activation of myosin light chain kinases (MLCK) may result in change of endothelial cell shape and opening of the intercellular gap with subsequent vascular leakage. Male C57Bl6 mice were subjected to endovascular perforation. Brain water content was determined by wet-dry ratio and BBB integrity by Evans-Blue extravasation. The specific MLCK inhibitor ML-7 was administered to the mice to determine the role of the contractile apparatus of the neurovascular unit in determining brain water content, BBB integrity, neurofunctional outcome, brain damage, and survival at 7 days after SAH. Inhibition of MLCK significantly reduced BBB permeability (Evans Blue extravasation - 28%) and significantly decreased edema formation in comparison with controls (- 2%). MLCK-treated mice showed reduced intracranial pressure (- 53%), improved neurological outcome at 24 h and 48 h after SAH, and reduced 7-day mortality. Tight junction proteins claudin-5 and zonula occludens-1 levels were not influenced by ML-7 at 24 h after insult. The effect of ML-7 on pMLC was confirmed in brain endothelial cell culture (bEnd.3 cells) subjected to 4-h oxygen-glucose deprivation. The present study indicates that MLCK contributes to blood-brain barrier dysfunction after SAH by a mechanism that does not involve modulation of tight junction protein levels, but via activation of the contractile apparatus of the endothelial cell skeleton. This underlying mechanism may be a promising target for the treatment of SAH.

ML-7 attenuates airway inflammation and remodeling via inhibiting the secretion of Th2 cytokines in mice model of asthma.

Previous studies have indicated that smooth muscle myosin light chain kinase (MLCK) has a prominent role in the regulation of smooth muscle contraction, which tends to be upregulated in asthma. In recent years, numerous studies have reported that MLCK is intimately connected with the immunoregulatory mechanism of T cells. The imbalance of T helper type 1 cells (Th1)/Th2 constitutes the immune­associated pathological basis of chronic asthma. Th2­associated cytokines, including interleukin­4, ­5, ­13, ­25 and ­33, are involved in airway inflammation, hyperresponsiveness and remodeling, which leads to a progressive decline in lung function. The purpose of the present study was to verify whether inhibition of bronchial MLCK attenuated the expression Th2­associated cytokines in asthmatic mice, including the above­mentioned ones. Female BALB/c mice were used to establish an ovalbumin (OVA)­induced model of asthma, of which one group was treated with the MLCK inhibitor (5-iodonaphthalene-1-sulfonyl) homopiperazine (ML­7). The inhibitor of MLCK, ML­7 attenuated airway inflammation and remodeling by reducing inflammatory cell infiltration and the secretion of Th2 cytokines in mice model of asthma, which may represent a promising therapeutic strategy for asthma.

Cytokinesis defect in BY-2 cells caused by ATP-competitive kinase inhibitors.

Cytokinesis is last but not least in cell division as it completes the formation of the two cells. The main role in cell plate orientation and expansion have been assigned to microtubules and kinesin proteins. However, recently we reported severe cytokinesis defect in BY-2 cells not accompanied by changes in microtubules dynamics. Here we also confirmed that distribution of kinesin NACK1 is not the cause of cytokinesis defect. We further explored inhibition of the cell plate expansion by ATP-competitive inhibitors. Two different inhibitors, 5-Iodotubercidin and ML-7 resulted in a very similar phenotype, which indicates that they target same protein cascade. Interestingly, in our previous study we showed that 5-Iodotubercidin treatment affects concentration of actin filaments on the cell plate, while ML-7 is inhibitor of myosin light chain kinase. Although not directly, it indicates importance of actomyosin complex in plant cytokinesis.

ML-7 amplifies the quinocetone-induced cell death through akt and MAPK-mediated apoptosis on HepG2 cell line.

The study aims at evaluating the combination of the quinocetone and the ML-7 in preclinical hepatocellular carcinoma models. To this end, the effect of quinocetone and ML-7 on apoptosis induction and signaling pathways was analyzed on HepG2 cell lines. Here, we report that ML-7, in a nontoxic concentration, sensitized the HepG2 cells to quinocetone-induced cytotoxicity. Also, ML-7 profoundly enhances quinocetone-induced apoptosis in HepG2 cell line. Mechanistic investigations revealed that ML-7 and quinocetone act in concert to trigger the cleavage of caspase-8 as well as Bax/Bcl-2 ratio up-regulation and subsequent cleavage of Bid, capsases-9 and -3. Importantly, ML-7 weakened the quinocetone-induced Akt pathway activation, but strengthened the phosphorylation of p-38, ERK and JNK. Further treatment of Akt activator and p-38 inhibitor almost completely abolished the ML-7/quinocetone-induced apoptosis. In contrast, the ERK and JNK inhibitor aggravated the ML-7/quinocetone-induced apoptosis, indicating that the synergism critically depended on p-38 phosphorylation and HepG2 cells provoke Akt, ERK and JNK signaling pathways to against apoptosis. In conclusion, the rational combination of quinocetone and ML-7 presents a promising approach to trigger apoptosis in hepatocellular carcinoma, which warrants further investigation.

Biphasic force response to iso-velocity stretch in airway smooth muscle.

Airway smooth muscle (ASM) in vivo is constantly subjected to oscillatory strain due to tidal breathing and deep inspirations. ASM contractility is known to be adversely affected by strains, especially those of large amplitudes. Based on the cross-bridge model of contraction, it is likely that strain impairs force generation by disrupting actomyosin cross-bridge interaction. There is also evidence that strain modulates muscle stiffness and force through induction of cytoskeletal remodeling. However, the molecular mechanism by which strain alters smooth muscle function is not entirely clear. Here, we examine the response of ASM to iso-velocity stretches to probe the components within the muscle preparation that give rise to different features in the force response. We found in ASM that force response to a ramp stretch showed a biphasic feature, with the initial phase associated with greater muscle stiffness compared with that in the later phase, and that the transition between the phases occurred at a critical strain of ∼3.3%. Only strains with amplitudes greater than the critical strain could lead to reduction in force and stiffness of the muscle in the subsequent stretches. The initial-phase stiffness was found to be linearly related to the degree of muscle activation, suggesting that the stiffness stems mainly from attached cross bridges. Both phases were affected by the degree of muscle activation and by inhibitors of myosin light-chain kinase, PKC, and Rho-kinase. Different responses due to different interventions suggest that cross-bridge and cytoskeletal stiffness is regulated differently by the kinases.

Myosin IIA is essential for Shigella flexneri cell-to-cell spread.

A key feature of Shigella pathogenesis is the ability to spread from cell-to-cell post-invasion. This is dependent on the bacteria's ability to initiate de novo F-actin tail polymerisation, followed by protrusion formation, uptake of bacteria-containing protrusion and finally, lysis of the double membrane vacuole in the adjacent cell. In epithelial cells, cytoskeletal tension is maintained by the actin-myosin II networks. In this study, the role of myosin II and its specific kinase, myosin light chain kinase (MLCK), during Shigella intercellular spreading was investigated in HeLa cells. Inhibition of MLCK and myosin II, as well as myosin IIA knockdown, significantly reduced Shigella plaque and infectious focus formation. Protrusion formation and intracellular bacterial growth was not affected. Low levels of myosin II were localised to the Shigella F-actin tail. HeLa cells were also infected with Shigella strains defective in cell-to-cell spreading. Unexpectedly loss of myosin IIA labelling was observed in HeLa cells infected with these mutant strains. This phenomenon was not observed with WT Shigella or with the less abundant myosin IIB isoform, suggesting a critical role for myosin IIA.

Cytotoxic Effects on HL-60 Cells of Myosin Light Chain Kinase Inhibitor ML-7 Alone and in Combination with Flavonoids.

Uncontrolled cell growth and increased cell proliferation are major features of cancer that are dependent on the stable structure and dynamics of the cytoskeleton. Since stable cytoskeleton structure and dynamics are partly regulated by myosin light chain kinase (MLCK), many current studies focused on MLCK inhibition as a chemotherapeutic target. As a potent and selective MLCK inhibitor, ML-7 [1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazapine hydrochloride] is a promising candidate for an anticancer agent, which would induce apoptosis as well as prevents invasion and metastasis in certain types of cancer cells. This study assessed cytotoxic effects of ML-7 against HL-60 cells and therapeutic efficacy of ML-7 as a potential antileukemia agent. Trypan-blue exclusion assays showed dose- and time- dependent decreases in ML-7 treated HL-60 cells (p < 0.05). Comet assays revealed a significant increase in DNA damage in HL-60 cells after treatment with 40 µM ML-7 for 2 h. Sub-G1 fractions, analyzed by flow cytometry increased in a dose-dependent manner, suggesting that ML-7 can induce apoptotic cell death in HL-60 cells. ML-7 was selectively cytotoxic towards HL-60 cells; not affecting normal human lymphocytes. That selective effect makes it a promising potential anti-leukemia agent. In addition, anticancer efficacy of ML-7 in combination with flavonoids (genistein or quercetin) or anticancer drugs (cisplatin or AraC) against HL-60 cells was assessed. Combination of ML-7 with flavonoids increased the anticancer effect of ML-7 to a greater extent than combination with the anticancer drugs. This implies that ML-7 in combination with flavonoids could increase the efficacy of anticancer treatment, while avoiding side effects cansed by conventional anticancer drug-containing combination chemotherapy.