Actin Research Articles

Calcium has a direct effect on thick filament activation in porcine myocardium.

Sarcomere activation in striated muscle requires both thin filament-based and thick filament-based activation mechanisms. Recent studies have shown that myosin heads on the thick filaments undergo OFF to ON structural transitions in response to calcium (Ca2+) in permeabilized porcine myocardium in the presence of a small molecule inhibitor that eliminated active force. The changes in X-ray diffraction signatures of OFF to ON transitions were interpreted as Ca2+ acting to activate the thick filaments. Alternatively, Ca2+ binding to troponin could initiate a Ca2+-dependent crosstalk from the thin filament to the thick filament via interfilament connections such as the myosin binding protein-C. Here, we exchanged native troponin in permeabilized porcine myocardium for troponin containing the cTnC D65A mutation, which disallows the activation of troponin through Ca2+ binding to determine if Ca2+-dependent thick filament activation persists in the absence of thin filament activation. After the exchange protocol, over 95% of the Ca2+-activated force was eliminated. Equatorial intensity ratio increased significantly in both WT and D65A exchanged myocardium with increasing Ca2+ concentration. The degree of helical ordering of the myosin heads decreased by the same amount in WT and D65A myocardium when Ca2+ concentration increased. These results are consistent with a direct effect of Ca2+ in activating the thick filament rather than an indirect effect due to Ca2+-mediated crosstalk between the thick and thin filaments.

Buoyancy Modes in a Low Entropy Bubble

AbstractIn the nightside region of Earth's magnetosphere, braking oscillations or buoyancy modes have been associated with the occurrence of low entropy bubbles. These bubbles form in the plasma sheet, particularly during geomagnetically disturbed times, and because of interchange, move rapidly earthward and may eventually come to rest in the inner plasma sheet or inner magnetosphere. Upon arrival, they often exhibit damped oscillations with periods of a few minutes and are associated with Pi2 pulsations. Previously we used the thin filament approximation to compare the frequencies and modes of buoyancy waves using magnetohydrodynamic (MHD) ballooning and classic interchange theory. Interchange oscillations differ from the more general MHD oscillations by assuming constant pressure along a magnetic field line. It was determined that MHD ballooning and interchange modes are similar for plasma sheet field lines but differ for field lines that map to the inner magnetosphere. This suggested that the classic interchange formulation was only valid in the plasma sheet. This paper tests the hypothesis that the agreement between MHD ballooning and classic interchange could be restored inside a bubble. We create a small region of entropy depletion in the magnetotail and compare the buoyancy mode properties. At some locations inside the bubble, the MHD ballooning buoyancy modes resemble interchange modes but with lower frequencies than those of the unperturbed background. Unstable modes are found on the earthward edge of the bubble, while at the tailward edge, MHD ballooning predicts a slow mode wave solution not seen in the pure interchange solution.

High-intensity exercise-related sudden cardiac death in hypertrophy cardiomyopathy

Abstract Background Whether exercise provokes sudden cardiac death (SCD) in patients with hypertrophic cardiomyopathy (HCM) is uncertain. Current European guidelines recommend against high-intensity exercise in individuals with high SCD risk or obstructive physiology. Purpose We investigated the factors associated with exercise-related SCD in HCM. Methods We retrospectively analyzed of 2,844 consecutive patients with HCM who underwent evaluation between 2005 and 2023 at a high-volume tertiary center. SCD events were defined as a composite of aborted SCD, appropriate implantable cardioverter-defibrillator (ICD) shock, and SCD. Among 80 first SCD events (2.8%), 75 events had a description of the physical activity at the time of the event. Patients were classified into two groups according to physical activity level at the time of the SCD event. Group 1 included patients with high-intensity exercise-related SCD events, defined as happening during or immediately after high-intensity exercise or physical activity, while Group 2 included patients with SCD events happening at rest or during light or moderate physical activity. Results The 75 first SCD events included 44 aborted SCD, 22 appropriate ICD shocks, and 9 SCD. Median age at SCD event was 54 (IQR 39.5–62.5) years and 60 (80%) were men. Patients in Group 1 (n=14) were younger than patients in Group 2 (n=61) (23.5 [IQR 16–48] vs. 55 [IQR 44–64] years, p<0.001), and most patients in group 1 (92.9%) were under the age of 60 years. Patients in Group 1 had slightly smaller left ventricular (LV) end-diastolic dimension and had higher ESC SCD risk scores (3.6 [IQR 2.5–6.7] vs. 2.4 [IQR 1.7–4.8], p=0.044) than patients in Group 2; however, there were no significant differences in the proportion of high-risk patients (28.6% vs. 14.8%, p=0.396) or patients with AHA major SCD risk factors (57.1% vs. 55.7%, p=0.999). Otherwise, there were no significant differences in the presence of obstructive physiology, apical HCM, and apical aneurysm, LV systolic and diastolic function, and comorbidities between the two groups. Furthermore, of the 29 patients who underwent genetic testing, there was no difference in the proportion of patients with pathogenic sarcomeric variants according to exercise-related SCD (50.0% vs. 65.2%, p=0.683). To note, there was a non-significant tendency towards higher proportion of thin filament variants in Group 1 than Group 2 among those with pathogenic sarcomeric variants (100% [3/3] vs 26.7% [4/15], p=0.084). Conclusions Most high-intensity exercise-related SCD events happened under the age of 60. The ESC SCD risk score was higher in patients with high-intensity exercise-related SCD events, although the SCD risk categories or AHA risk factors could not discriminate patients at higher risk of events. These results suggest that exercise recommendations should not be based on SCD risk category, presence of obstruction, or degree of LV hypertrophy.

Genetic variation, structural analysis, and virulence implications of BimA and BimC in clinical isolates of Burkholderia pseudomallei in Thailand

Melioidosis is a life-threatening tropical disease caused by an intracellular gram-negative bacterium Burkholderia pseudomallei. B. pseudomallei polymerizes the host cell actin through autotransporters, BimA, and BimC, to facilitate intracellular motility. Two variations of BimA in B. pseudomallei have been reported previously: BimABp and BimA B. mallei-like (BimABm). However, little is known about genetic sequence variations within BimA and BimC, and their potential effect on the virulence of B. pseudomallei. This study analyzed 1,294 genomes from clinical isolates of patients admitted to nine hospitals in northeast Thailand between 2015 and 2018 and performed 3D structural analysis and plaque-forming efficiency assay. The genomic analysis identified 10 BimABp and 5 major BimC types, in the dominant and non-dominant lineages of the B. pseudomallei population structure. Our protein prediction analysis of all BimABp and major BimC variants revealed that their 3D structures were conserved compared to those of B. pseudomallei K96243. Sixteen representative strains of the most distant BimABp types were tested for plaque formation and the development of polar actin tails in A549 epithelial cells. We found that all isolates retained these functions. These findings enhance our understanding of the prevalence of BimABp and BimC variants and their implications for B. pseudomallei virulence.

Distinct mechanisms drive divergent phenotypes in hypertrophic and dilated cardiomyopathy associated TPM1 variants.

Hypertrophic and dilated cardiomyopathies (HCM and DCM, respectively) are inherited disorders that may be caused by mutations to the same sarcomeric protein but have completely different clinical phenotypes. The precise mechanisms by which point mutations within the same gene bring about phenotypic diversity remain unclear. Our objective has been to develop a mechanistic explanation of diverging phenotypes in two TPM1 mutations, E62Q (HCM) and E54K (DCM). Drawing on data from the literature and experiments with stem cell-derived cardiomyocytes expressing the TPM1 mutations of interest, we constructed computational simulations that provide plausible explanations of the distinct muscle contractility caused by each variant. In E62Q, increased calcium sensitivity and hypercontractility was explained most accurately by a reduction in effective molecular stiffness of tropomyosin and alterations in its interactions with the actin thin filament that favor the 'closed' regulatory state. By contrast, the E54K mutation appeared to act via long-range allosteric interactions to increase the association rate of the C-terminal troponin I mobile domain to tropomyosin/actin. These mutation-linked molecular events produced diverging alterations in gene expression that can be observed in human engineered heart tissues. Modulators of myosin activity confirmed our proposed mechanisms by rescuing normal contractile behavior in accordance with predictions.

The D75N and P161S Mutations in the C0-C2 Fragment of cMyBP-C Associated with Hypertrophic Cardiomyopathy Disturb the Thin Filament Activation, Nucleotide Exchange in Myosin, and Actin-Myosin Interaction.

About half of the mutations that lead to hypertrophic cardiomyopathy (HCM) occur in the MYBPC3 gene. However, the molecular mechanisms of pathogenicity of point mutations in cardiac myosin-binding protein C (cMyBP-C) remain poorly understood. In this study, we examined the effects of the D75N and P161S substitutions in the C0 and C1 domains of cMyBP-C on the structural and functional properties of the C0-C1-m-C2 fragment (C0-C2). Differential scanning calorimetry revealed that these mutations disorder the tertiary structure of the C0-C2 molecule. Functionally, the D75N mutation reduced the maximum sliding velocity of regulated thin filaments in an in vitro motility assay, while the P161S mutation increased it. Both mutations significantly reduced the calcium sensitivity of the actin-myosin interaction and impaired thin filament activation by cross-bridges. D75N and P161S C0-C2 fragments substantially decreased the sliding velocity of the F-actin-tropomyosin filament. ADP dose-dependently reduced filament sliding velocity in the presence of WT and P161S fragments, but the velocity remained unchanged with the D75N fragment. We suppose that the D75N mutation alters nucleotide exchange kinetics by decreasing ADP affinity to the ATPase pocket and slowing the myosin cycle. Our molecular dynamics simulations mean that the D75N mutation affects myosin S1 function. Both mutations impair cardiac contractility by disrupting thin filament activation. The results offer new insights into the HCM pathogenesis caused by missense mutations in N-terminal domains of cMyBP-C, highlighting the distinct effects of D75N and P161S mutations on cardiac contractile function.

Effects of Cannabidiol on Intestinal Myofibroblast Fibrotic PathwaysPATHWAYS

Inflammatory bowel disease (IBD) includes two immune-mediated disorders, Crohn’s disease (CD) and ulcerative colitis (UC). IBD is characterized by recurrent and chronic inflammation of the gastrointestinal tract. IBD is lifelong, and there is no cure. There has been an interest in new advanced medications to decrease inflammation and manage symptoms. Many of these new medications are expensive and depress the immune system, leaving many patients looking for alternatives to current medical therapies. Some IBD patients use medical marijuana to relieve the symptoms of this disease, and cannabidiol (CBD) in particular shows potential for reducing inflammation. This study investigates the potential therapeutic effects of CBD on IBD by examining its impact on fibrotic pathways in human intestinal myofibroblast (InMyoFib) cells. Cultured InMyoFibs were pre-treated with CBD, followed by transforming growth factor-beta 1 (TGF-β1) to stimulate a pro-fibrotic phenotype and mimic human IBD in the lab. Expression of genes associated with fibrosis, inflammation, and cannabinoid signaling was measured by quantitative polymerase chain reaction (qPCR). Fibrotic protein expression in the culture media was analyzed using enzyme-linked immunosorbent assays (ELISA). CBD pre-treatment before TGF-β1 stimulation significantly reduced the expression of pro-fibrotic genes ACTA2, CCN2, COL1A1, and SERPINH1 compared to cells stimulated with TGF-β1 alone. Additionally, CBD increased the expression of inflammation-related genes IL6 and PTGS2 and the lipid metabolism gene PPARG, known to have anti-fibrotic properties. These findings suggest that CBD may modulate fibrotic and inflammatory signaling pathways, providing a potential therapeutic avenue for IBD treatment.

Lipoleiomyoma of the uterus in a woman of reproductive age (clinical case)

Uterine lipoleiomyoma is one of the variants of uterine leiomyoma, which is histologically represented by the presence of mature fat and smooth muscle cells. The relevance of the coverage of this clinical case is determined by the extreme rarity of the development of uterine lipoleiomyoma in women of reproductive age. The purpose of this work was to record this clinical case in the world statistics of lipoleiomyoma, to evaluate methods of diagnosis and differential diagnosis, to determine the role of early diagnosis of uterine lipoleioma in the subsequent tactics and scope of surgical treatment. Data from the medical records of a 41-year-old inpatient were analyzed. In addition to the generally accepted clinical and biochemical methods of blood and urine examination, electrocardiogram, ultrasound examination of the pelvic organs, the level of ovarian tumor markers in the blood (CA 125, HE4 and the ROMA index) was determined. The diagnosis of the disease was based on the data of pathohistological and immunohistochemical studies. Based on the results of a review of the medical literature, analysis of articles obtained as a result of a search of PubMed, SCOPUS, Web of Science, MedScape databases, the current state of the problem is highlighted, literary data related to the incidence, features of the clinical course, diagnosis and treatment of uterine lipoleiomyoma are summarized. The clinical case presented in the article demonstrates an incidental finding of a uterine lipoleiomyoma in a woman of reproductive age, measuring 30x25x20 cm, originated subserously from the body and cervix of the uterus, in the area of its isthmus. Under this condition, it occupied the entire Douglas space, the area of the sacro-uterine ligaments and the parietal peritoneum, intimately adjacent to the sigmoid and rectum, to the ureters and iliac vessels. The peculiarity of this clinical case is that sonographically uterine lipoleiomyoma was hidden under the "mask" of a dermoid cyst of the right ovary. Macroscopically, it differed from a typical lipoleiomyoma by the purple-bluish color of its outer surface and soot-colored, fine-lobed spongy structure on the section. The diagnosis of uterine lipoleiomyoma was verified only on the basis of pathohistological and immuno­histochemical research. Microscopically, the lipoleiomyoma had a mesenchymal structure with a pronounced vascular component and consisted of mitotically inactive bundles of smooth muscle cells and mature adipocytes. Im­munohistochemically, a positive reaction for caldesmon, desmin, smooth muscle actin alpha of tumor cells and for S.100 (DAKO, polyclonal) fatty cells was detected, which confirmed the hypothesis of direct transformation of smooth muscle cells existing in the leiomyoma of the uterus into fatty cells. This clinical case should complement the global statistical indicators of diagnosis of uterine lipoleiomyoma in women of reproductive age. Lipoleiomyoma should be considered as the primary diagnosis in case of detection of a large uterine tumor in women with excess body weight and be removed immediately after diagnosis, otherwise it is impossible to exclude its malignancy. For the planned diagnosis of neoplasms of the female genital organs, preference should be given to non-invasive research methods: magnetic resonance or computer tomography with contrast enhancement. The problem of these tumors lies in their unpredictable histogenesis, the unexpected presence of fat in the microscopic structure, and in the visual similarity to sarcomas. Verification of the diagnosis is carried out on the basis of pathohistological and immunohistochemical studies of the tumor preparation. Uterine lipoleioma can have a purplie-bluish color and develop by a broad base from the body and cervix of the uterus, as a result of "lipomatous" metaplasia of the uterine leiomyoma existing in a woman. Regular preventive examinations of women of all ages are crucial for timely detection of this rare neoplasm.

Combination of Green Tea, Green Coffee, and Turmeric Extract Improve the THOC5 and AIF1, but not ACTA2 and CNN1 Gene Expression in the Aortic Tissue of Metabolic Syndrome Model

Metabolic syndrome (MetS) is a group of risk factors in the form of central obesity, insulin resistance, dyslipidemia, and hypertension, increasing oxidative stress. This pathological event leads to the development of cardiovascular disease, for instance, atherosclerosis. Besides modifiable risk factors, non-modifiable risk factors such as genetic factors also play a role in the formation of atherosclerosis in MetS conditions such as THOC5, AIF1, CNN1, and ACTA2. Recently, natural compound derivatives, such as epigallocatechin-3-gallate (EGCG), chlorogenic acid (CGA), and turmeric, have shown beneficial effects in MetS improvement. This study aimed to investigate the effect of green tea, green coffee, and turmeric extract on the expression of THOC5, AIF1, CNN1, and ACTA2 genes that contributed to atherosclerotic vasculopathy development in the MetS rat model. Twenty-five MetS rat models were grouped into 4 groups (n = 5): Standard control (SC), MetS (MetS), a combination of green tea, green coffee, and turmeric extract with treatment doses: 300/100/150 mg/BW(C1) and 400/200/250 mg/BW(C2) group. The THOC5, AIF1, CNN1, and ACTA2 expression were measured at the end of treatment periods. This study found that administering green tea, green coffee, and turmeric extract can lower the expression of THOC5, AIF1, CNN1, and ACTA2. The correlation test showed that there is a strong correlation between THOC5 and AIF1 gene expression, with positive value. In summary, the combined effects of green tea, green coffee, and curcumin extract show significant promise as a potential anti-atherosclerosis treatment by improve the THOC5 and AIF1, but not ACTA2 and CNN1 gene expression in the aortic tissue of metabolic syndrome model. HIGHLIGHTS Metabolic syndrome is a group of risk factors consist of central obesity, insulin resistance, dyslipidemia, and hypertension. Individuals with MetS have an increased risk of developing atherosclerotic cardiovascular disease. Besides modifiable risk factors, non-modifiable risk factors such as genetic factors also play a role in the formation of atherosclerosis in MetS conditions such as THOC5, AIF1, CNN1, and ACTA2. This study aimed to investigate the effect of green tea, green coffee, and turmeric extract on the expression of THOC5, AIF1, CNN1, and ACTA2 genes that contributed to atherosclerotic vasculopathy development in the MetS rat model. Administering green tea, green coffee, and turmeric extract can lower the expression of THOC5, AIF1, CNN1, and ACTA2. The correlation test showed that there is a strong correlation between THOC5 and AIF1 gene expression, with positive value. In summary, the combined effects of green tea, green coffee, and curcumin extract show significant promise as a potential anti-atherosclerosis treatment by improve the THOC5 and AIF1, but not ACTA2 and CNN1 gene expression in the aortic tissue of metabolic syndrome model. GRAPHICAL ABSTRACT

Structural dynamics of the intrinsically disordered linker region of cardiac troponin T.

Troponin plays a central role in regulating heart contraction, and mutations in troponin can cause human cardiomyopathies. There are several functionally-significant regions of troponin that have not been structurally resolved, including the troponin T linker region that contains multiple cardiomyopathy mutations. In these unresolved regions, it is not possible to understand how changes in sequence affect function. We used computational and experimental techniques to demonstrate that this linker is dynamic and intrinsically disordered both in isolation and in the fully regulated thin filament. Moreover, we show how a cardiomyopathy mutation in this region affects function via allosteric disruption of intermolecular interactions. Our results highlight the need to consider how key mutations affect troponin disorder rather than the structure-function relationship.

Paraspinal muscle fibre structural and contractile characteristics demonstrate distinct irregularities in patients with spinal degeneration and deformity.

Paraspinal and spinopelvic muscular dysfunction are hypothesized to be a causative factor for spinal degeneration and deformity; however, our fundamental understanding of paraspinal muscle (dys)function remains limited. Twelve surgical patients with spinal degeneration were recruited and categorized into group DEG (four patients) with no sagittal imbalance and no usage of compensatory mechanisms; group DEG-COMP (four patients) with no sagittal imbalance through use of compensatory mechanisms; and group DEG-COMP-UNBAL (four patients) with sagittal imbalance despite use of compensatory mechanisms. From each patient, four biopsies were collected from right and left multifidus (MULT) and longissimus (LONG) for single fibre contractile and structural measurements. Eight of 48 (17%) biopsies did not exhibit any contractile properties. Specific force was not different between groups for the MULT (p = 0.47) but was greater in group DEG compared to group DEG-COMP-UNBAL for the LONG (p = 0.02). Force sarcomere-length properties were unusually variable both within and amongst patients in all groups. Thin filament (actin) lengths were in general shorter and more variable than published norms for human muscle. This study is the first to show a heightened intrinsic contractile muscle disorder (i.e. impaired specific force generation)in patients with spinal degeneration who are sagittally imbalanced (compared to patients without deformity). Additionally, there are clear indications that patients with spinal degeneration (all groups) have intrinsic force sarcomere-length properties that are dysregulated. This provides important insight into the pathophysiology of muscle weakness in this patient group.

Danicamtiv reduces myosin's working stroke but enhances contraction by activating the thin filament.

Heart failure is a leading cause of death worldwide, and there is a need to develop new treatments that improve outcomes for patients. Recently, the myosin-binding small molecule danicamtiv entered clinical trials for heart failure; however, its mechanism at the level of single myosin crossbridges is not well understood. We determined the molecular mechanism of danicamtiv and showed how drug-induced molecular changes can mechanistically increase heart contraction. Moreover, we demonstrate fundamental differences between danicamtiv and the related myosin-binding small molecule omecamtiv mecarbil that explain the improved diastolic function seen with danicamtiv. Our results have important implications for the design of new therapeutics for heart failure.

Astral-based DIA proteomics explored the flavor enhancement mechanism of Chinese traditional smoked bacon by staphylococcal co-fermentation

The proteolysis pattern during mixed fermentation of Staphylococcus cohnii WX-M8 and S. saprophyticus MY-A10 on Chinese bacon was still unknown. In this study, the changing laws of protein degradation products during staphylococcal mixed fermentation were analyzed, followed by an investigation of endogenous enzymes and cellular components, and finally an examination of flavor profiles. Results indicated that mixed fermentation improved protein degradation and promoted the production of peptides and free amino acids (FAAs). Proteolysis of S. saprophyticus MY-A10 was non-specific, and it promoted protein degradation by cooperating with cathepsin L1. S. cohnii WX-M8 was specific and acted mainly with calpain-3 in the thin filament. The fulfillment of S. cohnii WX-M8 function was enhanced in the presence of S. saprophyticus MY-A10. Mixed fermentation showed synergism with endogenous peptidases in degrading peptides to small-molecule peptides or FAAs and complementarity with endogenous dehydrogenases in converting FAAs to volatile organic compounds (VOCs).

Functional role of myosin-binding protein H in thick filaments of developing vertebrate fast-twitch skeletal muscle.

Myosin-binding protein H (MyBP-H) is a component of the vertebrate skeletal muscle sarcomere with sequence and domain homology to myosin-binding protein C (MyBP-C). Whereas skeletal muscle isoforms of MyBP-C (fMyBP-C, sMyBP-C) modulate muscle contractility via interactions with actin thin filaments and myosin motors within the muscle sarcomere "C-zone," MyBP-H has no known function. This is in part due to MyBP-H having limited expression in adult fast-twitch muscle and no known involvement in muscle disease. Quantitative proteomics reported here reveal that MyBP-H is highly expressed in prenatal rat fast-twitch muscles and larval zebrafish, suggesting a conserved role in muscle development and prompting studies to define its function. We take advantage of the genetic control of the zebrafish model and a combination of structural, functional, and biophysical techniques to interrogate the role of MyBP-H. Transgenic, FLAG-tagged MyBP-H or fMyBP-C both localize to the C-zones in larval myofibers, whereas genetic depletion of endogenous MyBP-H or fMyBP-C leads to increased accumulation of the other, suggesting competition for C-zone binding sites. Does MyBP-H modulate contractility in the C-zone? Globular domains critical to MyBP-C's modulatory functions are absent from MyBP-H, suggesting that MyBP-H may be functionally silent. However, our results suggest an active role. In vitro motility experiments indicate MyBP-H shares MyBP-C's capacity as a molecular "brake." These results provide new insights and raise questions about the role of the C-zone during muscle development.

Influence of native thin filament type on the regulation of atrial and ventricular myosin motor activity

Ca2+-mediated activation of thin filaments is a crucial step in initiating striated muscle contraction. To gain mechanistic insight into this regulatory process, thin filament (TF) components and myosin motors from diverse species and tissue sources are often combined in minimal in vitro systems. The contribution of tissue-specific TF composition with native myosin motors in generating contraction speed remains unclear. To examine TF-mediated regulation, we established a procedure to purify native TFs (nTF) and myosin motors (M-II) from the same cardiac tissue samples as low as 10 mg and investigated their influence on gliding speeds and Ca2+ sensitivity. The rabbit atrial and ventricular nTFs and M-II were assessed in in vitro nTF motility experiments under varying Ca2+ concentrations. The speed-pCa relationship yielded a maximum TF speed of 2.58 μm/s for atrial (aM-II) and 1.51 μm/s for ventricular myosin (vM-II), both higher than the respective unregulated actin filament gliding speeds. The Ca2+ sensitivity was different for both protein sources. After swapping the nTFs, the ventricular TFs increased their gliding speed on atrial myosin, while the atrial nTFs reduced their gliding speed on ventricular myosin. Swapping of the nTFs decreased the calcium sensitivity for both vM-II and aM-II, indicating a strong influence of the thin filament source. These studies suggest that the nTF-myosin combination is critical to understanding the Ca2+ sensitivity of the shortening speed. Our approach is highly relevant to studying precious human cardiac samples, i.e., small myectomy samples, to address the alteration of contraction speed and Ca2+ sensitivity in cardiomyopathies.

Cryo-EM structures of cardiac muscle α-actin mutants M305L and A331P give insights into the structural mechanisms of hypertrophic cardiomyopathy

Cardiac muscle α-actin is a key protein of the thin filament in the muscle sarcomere that, together with myosin thick filaments, produce force and contraction important for normal heart function. Missense mutations in cardiac muscle α-actin can cause hypertrophic cardiomyopathy, a complex disorder of the heart characterized by hypercontractility at the molecular scale that leads to diverse clinical phenotypes. While the clinical aspects of hypertrophic cardiomyopathy have been extensively studied, the molecular mechanisms of missense mutations in cardiac muscle α-actin that cause the disease remain largely elusive. Here we used cryo-electron microscopy to reveal the structures of hypertrophic cardiomyopathy-associated actin mutations M305L and A331P in the filamentous state. We show that the mutations have subtle impacts on the overall architecture of the actin filament with mutation-specific changes in the nucleotide binding cleft active site, interprotomer interfaces, and local changes around the mutation site. This suggests that structural changes induced by M305L and A331P have implications for the positioning of the thin filament protein tropomyosin and the interaction with myosin motors. Overall, this study supports a structural model whereby altered interactions between thick and thin filament proteins contribute to disease mechanisms in hypertrophic cardiomyopathy.

30P Nemaline myopathy-linked TNNT1 mutations are associated with aberrant thin filament extensibility and myofibre hyper-contractility

30P Nemaline myopathy-linked TNNT1 mutations are associated with aberrant thin filament extensibility and myofibre hyper-contractility

Preliminary antifibrotic and vasoconstrictor effects of adrenaline in Schlemm's canal and suprachoroidal minimally invasive glaucoma surgery in primary open-angle glaucoma.

To investigate the antifibrotic and vasoconstrictor effects of adrenaline in Schlemm's canal and suprachoroidal minimally invasive glaucoma surgery (MIGS). Human trabecular meshwork (TM) cells were treated with different concentrations of adrenaline (0%, 0.0005%, 0.01%), and we measured the effects on contractility, cell viability and the expression of key cell cycle and fibrosis genes. Adrenaline 0.05% was also injected intracamerally in five primary open-angle glaucoma patients undergoing iStent inject or MINIject surgery combined with phacoemulsification. All patients were assessed for ocular and systemic adverse reactions, including the effects on intraoperative pupil size, preoperative and postoperative visual acuity, intraocular pressure, and anterior segment OCT results. Adrenaline significantly reduced the contractility of TM cells in a dose-dependent manner (87.8%, 80.6%, 7.9% matrix contraction with adrenaline 0%, 0.0005%, 0.01%, respectively). Adrenaline did not exhibit any significant cytotoxicity even at high concentrations (P > 0.05). Adrenaline 0.01% significantly downregulated the expression of key cell cycle genes in the G2 and M phases, and also decreased the expression of MRTFB and ACTA2 genes (P < 0.05). Intracameral injections of adrenaline 0.05% in the five MIGS patients did not result in any ocular or systemic adverse effects. We recommend intracameral injections of adrenaline 0.05% as a cheap and safe drug to be used before MIGS insertion. Adrenaline decreases the risk of bleeding from the trabecular meshwork and also exhibits antifibrotic effects by arresting the cell cycle, thereby increasing the postoperative success rates in MIGS. What is known Fibrosis is the main cause of surgical failure in minimally invasive glaucoma surgery (MIGS). Mitomycin-C and 5-fluorouracil are too toxic to be used inside the eye. What is new Adrenaline reduced the contractility of trabecular meshwork cells and inhibited the expression of key cell cycle genes and fibrosis genes, without significant cytotoxicity. Intracameral injection of adrenaline 0.05% did not result in any ocular or systemic adverse reactions in MIGS patients.

Mycobacterial PE/PPE proteins function as “personal protective equipment” against host defenses

Mycobacterium tuberculosis (Mtb) is the deadliest bacterial infection worldwide, but many molecular details of how it interacts with the innate immune system remain obscure. In particular, although Mtb secretes a large number of putative effector proteins, a relatively small number have assigned functions in facilitating host-pathogen interactions. One particularly large family of secreted mycobacterial proteins that remains poorly understood is the PE/PPE proteins. Despite numerous lines of evidence for potential roles in virulence and in mediating host-pathogen interactions, only a small fraction of these 170+ proteins have been well characterized. However, this large family of proteins is likely key for understanding how Mtb subverts immune responses, manipulates host cell biology, and establishes a successful infection. Here, we highlight examples of PE/PPEs that have well-defined effects on cell intrinsic pathways in macrophages during mycobacterial infection. Examples include PPE2, which blunts production of reactive oxygen species and nitric oxide; PE_PGRS33, which facilitates bacterial uptake; PE_PGRS29, which directly binds ubiquitin to promote host autophagy and limit pathologic inflammation; MirA, which facilitates actin tail formation to promote cell-to-cell spread; and others. Understanding the full spectrum of PE/PPE functions is critical for understanding Mtb pathogenesis and for developing new strategies to combat the worldwide TB pandemic. Advancing the lagging research efforts characterizing this mysterious family of effector proteins is critical for the TB field.

Medin Induces Pro-Inflammatory Activation of Human Brain Vascular Smooth Muscle Cells.

Medin is one of the most common amyloidogenic proteins and accumulates in the vasculature with aging. Vascular medin accumulation is associated with Alzheimer's disease, vascular dementia and aortic aneurysms. Medin impairs smooth muscle-dependent vasodilation in isolated human brain cerebral arteries. The role of medin in vascular smooth muscle (VSMC) activation is unknown. We aim to evaluate the effects of medin on human brain VSMC activation. VSMCs were exposed to physiologic doses of medin (0.5, 1 and 5 µM) without or with small molecule nuclear factor-κB (NFκB) inhibitor RO106-9920 (10 µM) for 20 hours. Polymerase chain reaction, Western blot/enzyme-linked immunosorbent assay were used to quantify gene and protein expressions/secretions, respectively, of pro-inflammatory factors (interleukin (IL)-6, IL-8 and monocyte chemoattractant protein (MCP)-1) and structural and enzyme proteins associated with VSMC phenotypic transformation (smooth muscle actin alpha 2 (ACTA2), myosin heavy chain 11 (MYH11) and NADPH oxidase 4 (NOX4)). Medin exposure increased VSMC gene expression and protein secretion of IL-6, IL-8 and MCP-1 (protein secretion 46.0±12.8x, 20.2±4.1x and 8.7±3.1x, respectively, medin 5 µM versus vehicle, all p<0.05). There was no change in gene or protein expressions of ACTA2, MYH11 and NOX4. Co-treatment with RO106-9920 reduced medin-induced increases in IL-6 and IL-8 and a trend towards reduced MCP-1 secretion. Medin induced pro-inflammatory activation of human brain VSMCs that is mediated, at least in part, by NFκB. Acute medin treatment did not alter structural proteins involved in VSMC phenotypic transformation. The findings support medin as a potential novel mediator of and therapeutic target for vascular aging pathology.