Regulatory Light Chain Phosphorylation Research Articles

Impact and mechanisms of drag-reducing polymers on shear stress regulation in pulmonary hypertension.

Pulmonary hypertension (PH) is a refractory disease characterized by elevated pulmonary artery pressure and resistance. Drag-reducing polymers (DRPs) are blood-soluble macromolecules that reduce vascular resistance by altering the blood dynamics and rheology. Our previous work indicated that polyethylene oxide (PEO) can significantly reduce the medial wall thickness and vascular resistance of the pulmonary arteries, but the specific mechanism is still unclear. This study was designed to investigate the role and mechanism of PEO on intracellular calcium [Ca2+] i and cytoskeletal proteins of endothelial cells (ECs) induced by low shear stress (LSS) in PH. Primary Pulmonary Artery Endothelial Cells (PAECs) were subjected to steady LSS (1 dyn/cm2) or physiological shear stress (SS) (10 dyn/cm2) for 20 h in a BioFlux 200 flow system. Calcium influx assays were conducted to evaluate the mechanisms of PEO on [Ca2+] i. Subsequently, taking the key protein that induces cytoskeletal remodeling, the regulatory light chain (RLC) phosphorylation, as the breakthrough point, this study focused on the two key pathways of PEO that regulate phosphorylation of RLC: Myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK) pathways. Our current research revealed that PEO at LSS (1 dyn/cm2) significantly suppressed LSS-induced [Ca2+] i and the expression level of transient receptor potential channel 1(TRPC1). In addition, ECs convert LSS stimuli into the upregulation of cytoskeletal proteins, including filamentous actin (F-actin), MLCK, ROCK, p-RLC, and pp-RLC. Further experiments using pharmacological inhibitors demonstrated that PEO at the LSS downregulated cytoskeleton-related proteins mainly through the ROCK and MLCK pathways. This study considered intracellular calcium and cytoskeleton rearrangement as entry points to study the application of PEO in the biomedical field, which has important theoretical significance and practical application value for the treatment of PH.

The structural and functional effects of myosin regulatory light chain phosphorylation are amplified by increases in sarcomere length and [Ca2+

Precise regulation of sarcomeric contraction is essential for normal cardiac function. The heart must generate sufficient force to pump blood throughout the body, but either inadequate or excessive force can lead to dysregulation and disease. Myosin regulatory light chain (RLC) is a thick-filament protein that binds to the neck of the myosin heavy chain. Post-translational phosphorylation of RLC (RLC-P) by myosin light chain kinase is known to influence acto-myosin interactions, thereby increasing force production and Ca2+-sensitivity of contraction. Here, we investigated the role of RLC-P on cardiac structure and function as sarcomere length and [Ca2+] were altered. We found that at low, non-activating levels of Ca2+, RLC-P contributed to myosin head disorder, though there were no effects on isometric stress production and viscoelastic stiffness. With increases in sarcomere length and Ca2+-activation, the structural changes due to RLC-P become greater, which translates into greater force production, greater viscoelastic stiffness, slowed myosin detachment rates and altered nucleotide handling. Altogether, these data suggest that RLC-P may alter thick-filament structure by releasing ordered, off-state myosin. These more disordered myosin heads are available to bind actin, which could result in greater force production as Ca2+ levels increase. However, prolonged cross-bridge attachment duration due to slower ADP release could delay relaxation long enough to enable cross-bridge rebinding. Together, this work further elucidates the effects of RLC-P in regulating muscle function, thereby promoting a better understanding of thick-filament regulatory contributions to cardiac function in health and disease. KEY POINTS: Myosin regulatory light chain (RLC) is a thick-filament protein in the cardiac sarcomere that can be phosphorylated (RLC-P), and changes in RLC-P are associated with cardiac dysfunction and disease. This study assesses how RLC-P alters cardiac muscle structure and function at different sarcomere lengths and calcium concentrations. At low, non-activating levels of Ca2+, RLC-P contributed to myofilament disorder, though there were no effects on isometric stress production and viscoelastic stiffness. With increases in sarcomere length and Ca2+-activation, the structural changes due to RLC-P become greater, which translates into greater force production, greater viscoelastic stiffness, slower myosin detachment rate and altered cross-bridge nucleotide handling rates. This work elucidates the role of RLC-P in regulating muscle function and facilitates understanding of thick-filament regulatory protein contributions to cardiac function in health and disease.

Fustin suppressed melanoma cell growth via cAMP/PKA-dependent mechanism.

Melanoma, a cancer arising from melanocytes, requires a novel treatment strategy because of the ineffectiveness of conventional therapies in certain patients. Fustin is a flavanonol found in young fustic (Cotinus coggygria). However, little is known about its antimelanoma effects. Our study demonstrates that fustin suppresses the growth of B16 melanoma cells. Phalloidin staining of cytoskeletal actin revealed that fustin induced a conformational change in the actin structure of melanoma cells, accompanied by suppressed phosphorylation of myosin regulatory light chain 2 (MLC2), a regulator of actin structure. Furthermore, the protein kinase A (cAMP-dependent protein kinase) inhibitor H89 completely attenuated fustin-induced downregulation of phosphorylated myosin phosphatase targeting subunit 1, which is involved in dephosphorylation of MLC2. In a mouse model, administration of fustin suppressed tumor growth in B16 melanoma cells without adverse effects. In conclusion, our findings suggest that fustin effectively suppresses melanoma cell growth both in vitro and in vivo.

Mechanistic basis for rescuing hypertrophic cardiomyopathy with myosin regulatory light chain phosphorylation.

We investigated the impact of the phosphomimetic (Ser15 → Asp15) myosin regulatory light chain (S15D-RLC) on the Super-Relaxed (SRX) state of myosin using previously characterized transgenic (Tg) S15D-D166V rescue mice, comparing them to the Hypertrophic Cardiomyopathy (HCM) Tg-D166V model and wild-type (WT) RLC mice. In the Tg-D166V model, we observed a disruption of the SRX state, resulting in a transition from SRX to DRX (Disordered Relaxed) state, which explains the hypercontractility of D166V-mutated myosin motors. The presence of the S15D moiety in Tg-S15D-D166V mice restored the SRX/DRX balance to levels comparable to Tg-WT, thus mitigating the hypercontractile behavior associated with the HCM-D166V mutation. Additionally, we investigated the impact of delivering the S15D-RLC molecule to the hearts of Tg-D166V mice via adeno-associated virus (AAV9) and compared their condition to AAV9-empty vector-injected or non-injected Tg-D166V animals. Tg-D166V mice injected with AAV9 S15D-RLC exhibited a significantly higher proportion of myosin heads in the SRX state compared to those injected with AAV9 empty vector or left non-injected. No significant effect was observed in Tg-WT hearts treated similarly. These findings suggest that AAV9-delivered phosphomimetic S15D-RLC modality mitigates the abnormal Tg-D166V phenotype without impacting the normal function of Tg-WT hearts. Global longitudinal strain analysis supported these observations, indicating that the S15D moiety can alleviate the HCM-D166V phenotype by restoring SRX stability and the SRX ↔ DRX equilibrium.

Ablation of skeletal muscle estrogen receptor alpha impairs contractility in male mice.

Estradiol and estrogen receptor α (ERα) have been shown to be important for the maintenance of skeletal muscle strength in females; however, little is known about the roles of estradiol and ERα in male muscle. The purpose of this study was to determine if skeletal muscle ERα is required for optimal contractility in male mice. We hypothesize that reduced ERα in skeletal muscle impairs contractility in male mice. Skeletal muscle-specific knockout (skmERαKO) male mice exhibited reduced strength across multiple muscles and several contractile parameters related to force generation and kinetics compared with wild-type littermates (skmERαWT). Isolated EDL muscle-specific isometric tetanic force, peak twitch force, peak concentric and peak eccentric forces, as well as the maximal rates of force development and relaxation were 11%-21% lower in skmERαKO compared with skmERαWT mice. In contrast, isolated soleus muscles from skmERαKO mice were not affected. In vivo peak torque of the anterior crural muscles was 20% lower in skmERαKO compared with skmERαWT mice. Muscle masses, contractile protein contents, fiber types, phosphorylation of the myosin regulatory light chain, and caffeine-elicited force did not differ between muscles of skmERαKO and skmERαWT mice, suggesting that strength deficits were not due to size, composition, or calcium release components of muscle contraction. These results indicate that in male mice, reduced skeletal muscle ERα blunts contractility to a magnitude similar to that previously reported in females; however, the mechanism may be sexually dimorphic.NEW & NOTEWORTHY We comprehensively measured in vitro and in vivo contractility of leg muscles with reduced estrogen receptor α (ERα) in male mice and reported that force generation and contraction kinetics are impaired. In contrast to findings in females, phosphorylation of myosin regulatory light chain cannot account for low force production in male skeletal muscle ERα knockout mice. These results indicate that ERα is required for optimal contractility in males and females but via sexually dimorphic means.

Drebrin is Required for Myosin-facilitated Actin Cytoskeletal Remodeling during Pulmonary Alveolar Development.

Alveolar septation increases gas-exchange surface area and requires coordinated cytoskeletal rearrangement in lung fibroblasts (LFs) to balance the demands of contraction and cell migration. We hypothesized that DBN (drebrin), a modulator of the actin cytoskeleton in neuronal dendrites, regulates the remodeling of the LF cytoskeleton. Using mice bearing a transgelin-Cre-targeted deletion of Dbn in pulmonary fibroblasts and pericytes, we examined alterations in alveolar septal outgrowth, LF spreading and migration, and actomyosin function. The alveolar surface area and number of alveoli were reduced, whereas alveolar ducts were enlarged, in mice bearing the dbn deletion (DBNΔ) compared with their littermates bearing only one dbn-Flox allele (control). Cultured DBNΔ LFs were deficient in their responses to substrate rigidity and migrated more slowly. Drebrin was abundant in the actin cortex and lamella, and the actin fiber orientation was less uniform in lamella of DBNΔ LFs, which limited the development of traction forces and altered focal adhesion dynamics. Actin fiber orientation is regulated by contractile NM2 (nonmuscle myosin-2) motors, which help arrange actin stress fibers into thick ventral actin stress fibers. Using fluorescence anisotropy, we observed regional intracellular differences in myosin regulatory light chain phosphorylation in control LFs that were altered by dbn deletion. Using perturbations to induce and then release stalling of NM2 on actin in LFs from both genotypes, we made predictions explaining how DBN interacts with actin and NM2. These studies provide new insight for diseases such as emphysema and pulmonary fibrosis, in which fibroblasts inappropriately respond to mechanical cues in their environment.

The MYPT2-regulated striated muscle-specific myosin light chain phosphatase limits cardiac myosin phosphorylation in vivo

The physiological importance of cardiac myosin regulatory light chain (RLC) phosphorylation by its dedicated cardiac myosin light chain kinase (cMLCK) has been established in both humans and mice. Constitutive RLC-phosphorylation, regulated by the balanced activities of cMLCK and myosin light chain phosphatase (MLCP), is fundamental to the biochemical and physiological properties of myofilaments. However, limited information is available on cardiac MLCP. In this study, we hypothesized that the striated muscle-specific MLCP regulatory subunit, MYPT2, targets the phosphatase catalytic subunit to cardiac myosin, contributing to the maintenance of cardiac function in vivo through the regulation of RLC phosphorylation. To test this hypothesis, we generated a floxed-PPP1R12B mouse model crossed with a cardiac-specific Mer-Cre-Mer to conditionally ablate MYPT2 in adult cardiomyocytes. Immunofluorescence microscopy using the gene-ablated tissue as a control confirmed the localization of MYPT2 to regions where it overlaps with a subset of RLC. Biochemical analysis revealed an increase in RLC-phosphorylation in vivo. The loss of MYPT2 demonstrated significant protection against pressure overload-induced hypertrophy, as evidenced by heart weight, qPCR of hypertrophy-associated genes, measurements of myocyte diameters, and expression of β-MHC protein. Furthermore, mantATP chase assays revealed an increased ratio of myosin heads distributed to the interfilament space in MYPT2-ablated heart muscle fibers, confirming that RLC-phosphorylation regulated by MLCP, enhances cardiac performance in vivo. Our findings establish MYPT2 as the regulatory subunit of cardiac MLCP, distinct from the ubiquitously expressed canonical smooth muscle MLCP. Targeting MYPT2 to increase cardiac RLC-phosphorylation in vivo may improve baseline cardiac performance, thereby attenuating pathological hypertrophy.

Integrin-Dependent Transient Density Increase in Detergent-Resistant Membrane Rafts in Platelets Activated by Thrombin.

Platelet lipid rafts are critical membrane domains for adhesion, aggregation, and clot retraction. Lipid rafts are isolated as a detergent-resistant membrane fraction via sucrose density gradient centrifugation. The platelet detergent-resistant membrane shifted to a higher density on the sucrose density gradient upon thrombin stimulation. The shift peaked at 1 min and returned to the control level at 60 min. During this time, platelets underwent clot retraction and spreading on a fibronectin-coated glass strip. Thrombin induced the transient tyrosine phosphorylation of several proteins in the detergent-resistant membrane raft fraction and the transient translocation of fibrin and myosin to the detergent-resistant membrane raft fraction. The level of phosphatidylserine (36:1) was increased and the level of phosphatidylserine (38:4) was decreased in the detergent-resistant membrane raft fraction via the thrombin stimulation. Furthermore, Glanzmann's thrombasthenia integrin αIIbβ3-deficient platelets underwent no detergent-resistant membrane shift to a higher density upon thrombin stimulation. As the phosphorylation of the myosin regulatory light chain on Ser19 was at a high level in Glanzmann's thrombasthenia resting platelets, thrombin caused no further phosphorylation of the myosin regulatory light chain on Ser19 or clot retraction. These observations suggest that the fibrin-integrin αIIbβ3-myosin axis and compositional change of phosphatidylserine species may be required for the platelet detergent-resistant membrane shift to a higher density upon stimulation with thrombin.

Long-term experimental hyperglycemia does not impair macrovascular endothelial barrier integrity and function in vitro

Hyperglycemia is a hallmark of type 2 diabetes implicated in vascular endothelial dysfunction and cardiovascular complications. Many in vitro studies identified endothelial apoptosis as an early outcome of experimentally modeled hyperglycemia emphasizing cell demise as a significant factor of vascular injury. However, endothelial apoptosis has not been observed in vivo until the late stages of type 2 diabetes. Here, we studied the long-term (up to 4 weeks) effects of high glucose (HG, 30 mM) on human umbilical vein endothelial cells (HUVEC) in vitro. HG did not alter HUVEC monolayer morphology, ROS levels, NO production, and exerted minor effects on the HUVEC apoptosis markers. The barrier responses to various clues were indistinguishable from those by cells cultured in physiological glucose (5 mM). Tackling the key regulators of cytoskeletal contractility and endothelial barrier revealed no differences in the histamine-induced intracellular Ca2+ responses, nor in phosphorylation of myosin regulatory light chain or myosin light chain phosphatase. Altogether, these findings suggest that vascular endothelial cells may well tolerate HG for relatively long exposures and warrant further studies to explore mechanisms involved in vascular damage in advanced type 2 diabetes.

Regulatory Light-Chain Phosphorylation During Weightlifting Training: Association With Postactivation Performance Enhancement.

Chiu, LZF, Fry, AC, Galpin, AJ, Salem, GJ, and Cabarkapa, D. Regulatory light-chain phosphorylation during weightlifting training: association with postactivation performance enhancement. J Strength Cond Res 37(10): e563-e568, 2023-Postactivation performance enhancement has been reported for multijoint resistance exercise, with both neural and intrinsic muscle mechanisms suggested as contributing factors. The purpose of this investigation was to examine whether regulatory light-chain (RLC) phosphorylation in a primary mover is associated with enhanced weightlifting performance. Nine male athletes performed 15 sets of 3 repetitions of a multijoint weightlifting activity (clean pull) at 85% 1 repetition maximum. Measures of performance, peak barbell velocity (PV), and average barbell power (AP) were determined by video analysis. Muscle biopsies were taken within 30-60 seconds of completion of the previous lifting set from the vastus lateralis before (PRE), during (MID), and after (POST) a training session. AP was significantly greater for sets 3, 4, and 5 compared with set 1, with large effect sizes (0.8-1.0). Increases in PV did not reach significance; however, the effect size increase for sets 3 and 4 versus set 1 was moderate (0.4). Relative change scores for AP and RLC phosphorylation were positively and negatively correlated at MID (r = 0.60; p = 0.05) and POST (r = -0.74; p = 0.01) exercise, respectively. These data suggest that RLC phosphorylation initially may be associated with postactivation performance enhancement during repeated multijoint exercise.

B-308 Focal Adhesion Kinase Activation is Involved in Tension Development During Contractile Stimulation of Mouse Detrusor Smooth Muscle to Modulate Bladder Dysfunction-Related Lower Urinary Tract Symptoms

Abstract Background The function of the bladder is to store and expel urine. Bladder dysfunction-related lower urinary tract symptoms (LUTS) is a common urological problem and negatively affects health-related quality of life. Investigation of detrusor muscle contraction mechanisms is necessary to understand its underlying pathophysiology. Voiding occurs through bladder muscle contraction, which is mediated primarily by the increased intracellular Ca2+ concentration-induced myosin regulatory light chain (MLC) phosphorylation. Recent studies showed that focal adhesion kinase (FAK)-regulated focal adhesion assembly and actin polymerization are involved in vascular and airway smooth muscle contraction. However, the role of FAK activation in visceral smooth muscle contraction is not well documented. This study aims to determine if FAK activation is involved in mouse detrusor smooth muscle contraction. Methods Normal adult male and female mouse bladders were harvested. The trigone and dome areas were cut, and the bladder body was used. The mucosa was carefully removed. Detrusor muscle strips (1.5–2 × 8–10 mm) were prepared and then suspended in a 20 mL tissue bath filled with physiological Krebs solution and bubbled with 95% O2 and 5% CO2 to obtain a pH of 7.4 at 37 °C. The isometric contraction was recorded. After establishing a stable baseline, the strips were stimulated with 120 mM potassium chloride (KCl), which was used to normalize the contractility induced by subsequent stimuli. One of the following specific inhibitors or the same volume of vehicle (DMSO) was applied and incubated for 30 min: PF-573228 (2 µM, a FAK inhibitor) or latrunculin B (1 µM, an inhibitor of actin polymerization). Then, the contractile responses to KCl (90 mM), electrical field stimulation (EFS, 2–32 Hz), or carbachol (CCh, 10−7.5–10−4.5 M ) were measured. In a separate experiment, the detrusor strips were stimulated with CCh (10 µM) after incubation with PF-573228 (2 µM) or vehicle (DMSO) for 30 min. Once the peak contraction was reached, the strips were snap-frozen for immunoblotting analysis of the phosphorylated FAK (p-FAK) and MLC (p-MLC). Statistical analysis was performed (GraphPad Prism 6). The contractile responses to KCl and EFS between vehicle and inhibitor groups were compared using a paired t-test. Nonlinear regression was used to compare CCh-induced dose-response curves. For the expression of phosphorylated proteins, one-way ANOVA, followed by Tukey’s posthoc test, was used for analysis. Results KCl-induced contractile responses decreased significantly after incubation with PF-573228 or latrunculin B compared to the corresponding vehicle group (P < 0.0001). The contractile responses induced by EFS were significantly inhibited by preincubation with PF-573228 at 8, 16, and 32 Hz (P < 0.05) or latrunculin B at 16 and 32 Hz (P < 0.01). Following the application of PF-573228 or latrunculin B, CCh-induced dose-response contractions were significantly lower than those in the corresponding vehicle-treated strips (P = 0.0021 and 0.0003, respectively). Immunoblotting analysis demonstrated that CCh stimulation caused increased expression of p-FAK and p-MLC, whereas preincubation with PF-573228 led to decreased expression of p-FAK but not p-MLC Conclusion FAK activation is involved in tension development induced by contractile stimulation in mouse detrusor muscle, probably through enhancing actin polymerization, but not MLC phosphorylation.

Multi-omics approaches revealed the therapeutic mechanisms of Suo-Quan-Wan for treating overactive bladder in spontaneously hypertensive rats

Ethnopharmacological relevanceSuo-Quan-Wan (SQW), a traditional Chinese prescription, has been used for hundreds of years to alleviate overactive bladder (OAB) symptoms such as frequent and nocturnal urination. However, limited modern research on OAB therapeutic targets has hindered the use and development of SQW. Aim of the studyThis study aimed to investigate the biological mechanisms and key targets of SQW on OAB in spontaneously hypertensive rats (SHR) using an integrated analysis of network pharmacology, transcriptome and metabolome. MethodsRats were divided into five groups: model group (SHR), control group (WKY), darifenacin group, high dose (SQWH) and low dose (SQWL) group. Urodynamic parameters and histological examination were detected. Network pharmacology, transcriptome, and metabolome were used to screen for disease gene targets, differential mRNA, and differential metabolites, respectively. The biological targets and mechanisms of SQW for OAB were analyzed. Western blotting was performed to verify the proteins of key differential targets. ResultsUrodynamics revealed a significant decrease in storage parameters in SHR. After SQW treatment, the inter-contraction interval, voided volume and bladder capacity increased by 2–3 times, as well as bladder compliance. Additionally, SQW improved the pathological changes in the urinary tract epithelium and the detrusor layer of the bladder in SHR. Metabolomic results showed an increase in arachidonic acid (AA) and cyclic adenosine monophosphate (cAMP) in plasma, suggesting the involvement of arachidonic acid metabolism and purine metabolism in SQW treatment. The downregulation of cytochrome P450 1B1 (CYP1B1), thromboxane-A synthase (TBXAS1), polyunsaturated fatty acid 5-lipoxygenase (ALOX5), and cAMP-specific 3′,5′-cyclic phosphodiesterase 4B (PDE4B) were confirmed through topological analysis and Venn analysis of omics data and network pharmacology. These proteins affected the metabolism of AA and cAMP, respectively, and consequently affected downstream proteins, such as transient receptor potential (TRP) cation channel proteins (e.g. TRPV1, TRPA1, and TRPM8), myosin light chain kinase (MLCK), and the phosphorylation of myosin regulatory light chain (p-MLC). ConclusionThis study initially elucidated the importance of AA and cAMP in the treatment of SQW, indicating the AA-CYP1B1/TBXAS1/ALOX5-TRPA1/TRPV1/TRPM8 and cAMP-PDE4B-MLCK-p-MLC pathways as the important pathways in SQW-treated SHR bladder in vivo.

The weakness of senescent dermal fibroblasts

Skin is the largest human organ with easily noticeable biophysical manifestations of aging. As human tissues age, there is chronological accumulation of biophysical changes due to internal and environmental factors. Skin aging leads to decreased elasticity and the loss of dermal matrix integrity via degradation. The mechanical properties of the dermal matrix are maintained by fibroblasts, which undergo replicative aging and may reach senescence. While the secretory phenotype of senescent fibroblasts is well studied, little is known about changes in the fibroblasts biophysical phenotype. Therefore, we compare biophysical properties of young versus proliferatively aged primary fibroblasts via fluorescence and traction force microscopy, single-cell atomic force spectroscopy, microfluidics, and microrheology of the cytoskeleton. Results show senescent fibroblasts have decreased cytoskeletal tension and myosin II regulatory light chain phosphorylation, in addition to significant loss of traction force. The alteration of cellular forces is harmful to extracellular matrix homeostasis, while decreased cytoskeletal tension can amplify epigenetic changes involved in senescence. Further exploration and detection of these mechanical phenomena provide possibilities for previously unexplored pharmaceutical targets against aging.

Divergence of cytokinesis and dimorphism control by myosin II regulatory light chain in fission yeasts

Non-muscle myosin II activation by regulatory light chain (Rlc1Sp) phosphorylation at Ser35 is crucial for cytokinesis during respiration in the fission yeast Schizosaccharomyces pombe. We show that in the early divergent and dimorphic fission yeast S.japonicus non-phosphorylated Rlc1Sj regulates the activity of Myo2Sj and Myp2Sj heavy chains during cytokinesis. Intriguingly, Rlc1Sj-Myo2Sj nodes delay yeast to hyphae onset but are essential for mycelial development. Structure-function analysis revealed that phosphorylation-induced folding of Rlc1Sp α1 helix into an open conformation allows precise regulation of Myo2Sp during cytokinesis. Consistently, inclusion of bulky tryptophan residues in the adjacent α5 helix triggered Rlc1Sp shift and supported cytokinesis in absence of Ser35 phosphorylation. Remarkably, unphosphorylated Rlc1Sj lacking the α1 helix was competent to regulate S.pombe cytokinesis during respiration. Hence, early diversification resulted in two efficient phosphorylation-independent and -dependent modes of Rlc1 regulation of myosin II activity in fission yeasts, the latter being conserved through evolution.

Long-Term Experimental Hyperglycemia Does Not Impair Macrovascular Endothelial Barrier Integrity and Function in vitro.

Hyperglycemia is a hallmark of type 2 diabetes implicated in vascular endothelial dysfunction and cardiovascular complications. Many in vitro studies identified endothelial apoptosis as an early outcome of experimentally modeled hyperglycemia emphasizing cell demise as a significant factor of vascular injury. However, endothelial apoptosis has not been observed in vivo until the late stages of type 2 diabetes. Here, we studied the long-term (up to 4 weeks) effects of high glucose (HG, 30 mM) on human umbilical vein endothelial cells (HUVEC) in vitro. HG did not alter HUVEC monolayer morphology, ROS levels, NO production, and exerted minor effects on the HUVEC apoptosis markers. The barrier responses to various clues were indistinguishable from those by cells cultured in physiological glucose (5 mM). Tackling the key regulators of cytoskeletal contractility and endothelial barrier revealed no differences in the histamine-induced intracellular Ca2+ responses, nor in phosphorylation of myosin regulatory light chain or myosin light chain phosphatase. Altogether, these findings suggest that vascular endothelial cells may well tolerate HG for relatively long exposures and warrant further studies to explore mechanisms involved in vascular damage in advanced type 2 diabetes.

ADT-OH improves intestinal barrier function and remodels the gut microbiota in DSS-induced colitis.

Owing to the increasing incidence and prevalence of inflammatory bowel disease (IBD) worldwide, effective and safe treatments for IBD are urgently needed. Hydrogen sulfide (H2S) is an endogenous gasotransmitter and plays an important role in inflammation. To date, H2S-releasing agents are viewed as potential anti-inflammatory drugs. The slow-releasing H2S donor 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH), known as a potent therapeutic with chemopreventive and cytoprotective properties, has received attention recently. Here, we reported its anti-inflammatory effects on dextran sodium sulfate (DSS)-induced acute (7 days) and chronic (30 days) colitis. We found that ADT-OH effectively reduced the DSS-colitis clinical score and reversed the inflammation-induced shortening of colon length. Moreover, ADT-OH reduced intestinal inflammation by suppressing the nuclear factor kappa-B pathway. In vivo and in vitro results showed that ADT-OH decreased intestinal permeability by increasing the expression of zonula occludens-1 and occludin and blocking increases in myosin II regulatory light chain phosphorylation and epithelial myosin light chain kinase protein expression levels. In addition, ADT-OH restored intestinal microbiota dysbiosis characterized by the significantly increased abundance of Muribaculaceae and Alistipes and markedly decreased abundance of Helicobacter, Mucispirillum, Parasutterella, and Desulfovibrio. Transplanting ADT-OH-modulated microbiota can alleviate DSS-induced colitis and negatively regulate the expression of local and systemic proinflammatory cytokines. Collectively, ADT-OH is safe without any short-term (5 days) or long-term (30 days) toxicological adverse effects and can be used as an alternative therapeutic agent for IBD treatment.

Restoration of Cardiac Myosin Light Chain Kinase Ameliorates Systolic Dysfunction by Reducing Superrelaxed Myosin.

Cardiac-specific myosin light chain kinase (cMLCK), encoded by MYLK3, regulates cardiac contractility through phosphorylation of ventricular myosin regulatory light chain. However, the pathophysiological and therapeutic implications of cMLCK in human heart failure remain unclear. We aimed to investigate whether cMLCK dysregulation causes cardiac dysfunction and whether the restoration of cMLCK could be a novel myotropic therapy for systolic heart failure. We generated the knock-in mice (Mylk3+/fs and Mylk3fs/fs) with a familial dilated cardiomyopathy-associated MYLK3 frameshift mutation (MYLK3+/fs) that had been identified previously by us (c.1951-1G>T; p.P639Vfs*15) and the human induced pluripotent stem cell-derived cardiomyocytes from the carrier of the mutation. We also developed a new small-molecule activator of cMLCK (LEUO-1154). Both mice (Mylk3+/fs and Mylk3fs/fs) showed reduced cMLCK expression due to nonsense-mediated messenger RNA decay, reduced MLC2v (ventricular myosin regulatory light chain) phosphorylation in the myocardium, and systolic dysfunction in a cMLCK dose-dependent manner. Consistent with this result, myocardium from the mutant mice showed an increased ratio of cardiac superrelaxation/disordered relaxation states that may contribute to impaired cardiac contractility. The phenotypes observed in the knock-in mice were rescued by cMLCK replenishment through the AAV9_MYLK3 vector. Human induced pluripotent stem cell-derived cardiomyocytes with MYLK3+/fs mutation reduced cMLCK expression by 50% and contractile dysfunction, accompanied by an increased superrelaxation/disordered relaxation ratio. CRISPR-mediated gene correction, or cMLCK replenishment by AAV9_MYLK3 vector, successfully recovered cMLCK expression, the superrelaxation/disordered relaxation ratio, and contractile dysfunction. LEUO-1154 increased human cMLCK activity ≈2-fold in the Vmax for ventricular myosin regulatory light chain phosphorylation without affecting the Km. LEUO-1154 treatment of human induced pluripotent stem cell-derived cardiomyocytes with MYLK3+/fs mutation restored the ventricular myosin regulatory light chain phosphorylation level and superrelaxation/disordered relaxation ratio and improved cardiac contractility without affecting calcium transients, indicating that the cMLCK activator acts as a myotrope. Finally, human myocardium from advanced heart failure with a wide variety of causes had a significantly lower MYLK3/PPP1R12B messenger RNA expression ratio than control hearts, suggesting an altered balance between myosin regulatory light chain kinase and phosphatase in the failing myocardium, irrespective of the causes. cMLCK dysregulation contributes to the development of cardiac systolic dysfunction in humans. Our strategy to restore cMLCK activity could form the basis of a novel myotropic therapy for advanced systolic heart failure.

Abstract 4248: Epithelial ovarian cancer causes cardiac dysfunction and myofilament dysregulation

Abstract Introduction: The intersections between cancer and cardiovascular disease (CVD) have become increasingly relevant in the care of both patient populations. To date, the cardio-oncology field has largely focused on how cancer treatment influences long-term cardiac health. Indeed, many cancer therapeutics show various toxic effects to the cardiovascular system. However, studies have also shown cardiac deficits in therapy-naïve cancer patients. This suggests that these two disease states interact through similar signaling pathways, yet the mechanisms underlying cancer-induced cardiac dysfunction remain unknown. Thus, we investigated cardiac structure and function throughout the progression of epithelial ovarian cancer (EOC). Methods: We used an orthotopic, syngeneic mouse model of ovarian cancer. Briefly, transformed murine ovarian surface epithelial cells from C57BL/6 mice (ID8; 1.0 × 106), or saline for surgical controls, were injected under the ovarian bursa of syngeneic mice. In this model, 60 days after tumor induction, mice form large ovarian masses, numerous peritoneal lesions, and develop abdominal ascites - consistent with the clinical features of stage III (advanced) EOC. To determine whether cardiac abnormalities precede the presence of advanced disease, at 45 days post-tumor induction, mice were anesthetized, and left ventricular (LV) structure and function were assessed by echocardiography and invasive hemodynamics, respectively. Myofilament activity was measured with an actomyosin MgATPase assay and protein phosphorylation was determined by SDS-PAGE with Pro-Q Diamond phosphoprotein staining. Results: EOC mice showed systolic (i.e., reduced contraction rate) and diastolic dysfunction (i.e., reduced relaxation rate, elevated diastolic filling pressure), with no evidence of hypertrophy or atrophy (i.e., no change in LV wall thickness or dimension). While there were no observed differences in myofilament ATPase activity, phosphorylation of regulatory myosin light chain 2 (MYL2) and cardiac myosin binding protein C (cMyBP-C) decreased, and tropomyosin (Tpm) phosphorylation increased in EOC mice. Conclusion: Together, these data demonstrate that ovarian cancer impairs cardiac function and alters the regulation of myofilament proteins critical for contraction and relaxation. Interestingly, the observed dephosphorylation of MYL2 and cMyBP-C and hyperphosphorylation of Tpm are consistent with features of heart failure. The findings build upon our current understanding of the interactions between cancer and CVD and show that deficits in the heart precede the development of advanced malignancy. Future work further will investigate the tumor-heart axis to identify how tumor development disrupts normal cardiac physiology. Citation Format: Leslie M. Ogilvie, Madison Pereira, Kathy Matuszewska, Bridget Coyle-Asbil, Luca J. Delfinis, Shivam Gandhi, Keith R. Brunt, Christopher G. Perry, W. Glen Pyle, Jim Petrik, Jeremy A. Simpson. Epithelial ovarian cancer causes cardiac dysfunction and myofilament dysregulation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4248.

Myosin II regulatory light chain phosphorylation and formin availability modulate cytokinesis upon changes in carbohydrate metabolism.

Cytokinesis, the separation of daughter cells at the end of mitosis, relies in animal cells on a contractile actomyosin ring (CAR) composed of actin and class II myosins, whose activity is strongly influenced by regulatory light chain (RLC) phosphorylation. However, in simple eukaryotes such as the fission yeast Schizosaccharomyces pombe, RLC phosphorylation appears dispensable for regulating CAR dynamics. We found that redundant phosphorylation at Ser35 of the S. pombe RLC homolog Rlc1 by the p21-activated kinases Pak1 and Pak2, modulates myosin II Myo2 activity and becomes essential for cytokinesis and cell growth during respiration. Previously, we showed that the stress-activated protein kinase pathway (SAPK) MAPK Sty1 controls fission yeast CAR integrity by downregulating formin For3 levels (Gómez-Gil et al., 2020). Here, we report that the reduced availability of formin For3-nucleated actin filaments for the CAR is the main reason for the required control of myosin II contractile activity by RLC phosphorylation during respiration-induced oxidative stress. Thus, the restoration of For3 levels by antioxidants overrides the control of myosin II function regulated by RLC phosphorylation, allowing cytokinesis and cell proliferation during respiration. Therefore, fine-tuned interplay between myosin II function through Rlc1 phosphorylation and environmentally controlled actin filament availability is critical for a successful cytokinesis in response to a switch to a respiratory carbohydrate metabolism.

Striated muscle MLCP is regulated by MYPT2 to limit baseline cardiac myosin phosphorylation.

Cardiac myosin is a hexameric protein comprised of two protamers that each have a heavy chain, a regulatory light chain (RLC), and an essential light chain. Constitutive phosphorylation of cardiac myosin in beating hearts at 0.4 mol phosphate/mol RLC is necessary for normal function. This level is maintained by balanced activities of the cardiac myosin light chain kinase (cMLCK) and myosin light chain phosphatase (MLCP). The canonical MLCP is well-studied in smooth muscles, and is defined as a trimeric holoenzyme comprised of a myosin target regulatory subunit MYPT1, a type-1 catalytic subunit PP1cβ, and an accessory protein M21. In striated muscles, the dominantly expressed regulatory subunit is MYPT2. There is limited information on the localization and regulatory contributions of MYPT2. Studies of regulation of cardiac myosin dephosphorylation is hindered by the fact that myosin dephosphorylation occurs rapidly in isolated cardiac myocytes, and cultured “cardiac” cell lines do not express the regulatory proteins at levels comparable to those of freshly isolated cardiac myocytes. We generated a floxed MYPT2 mouse line and conditionally knocked out nearly all detectable MYPT2 protein in cardiac myocytes, and used this model to determine that: 1) MYPT2 localizes to myosin in vivo, 2) MYPT1 expression does not compensate for the reduction in MYPT2 in the cardiac muscle, 3) the cardiac MLCP holoenzyme expression level is partly regulated by stabilization of the catalytic subunit by MYPT2, 4) without MYPT2-regulated MLCP activity, baseline cardiac RLC phosphorylation is increased to 0.6 mol phosphate/mol RLC, and 5) MYPT2 knockout animals have reduced responses to pressure-overload induced cardiac hypertrophy. These findings help to fill knowledge-gaps about the fundamental properties of the cardiac muscle MLCP.