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α sarcomeric actin

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α-sarcomeric actin is a protein found in the sarcomeres of skeletal and cardiac muscle cells. It is a key component of the contractile machinery responsible for muscle contraction. This protein plays a crucial role in the structural integrity and function of muscle tissues.

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Lab products found in correlation

Lsm 700 confocal microscope, by Zeiss (4 mentions) Fitc isolectin b4, by Vector Laboratories (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions) A1 confocal microscope, by Nikon (2 mentions) Lsm 700, by Zeiss (2 mentions) Apoptag peroxidase in situ oligo ligation kit, by Merck Group (1 mentions) In situ cell death detection kit, by Merck Group (1 mentions) Sudan black b, by Merck Group (1 mentions) Alexa fluor 488, by Thermo Fisher Scientific (1 mentions) Anti gfp staining, by Abcam (1 mentions) Bromodeoxyuridine (brdu), by Roche (1 mentions) Myosin heavy chain, by Merck Group (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Vitamin b12, by Merck Group (1 mentions) Penicillin, by Merck Group (1 mentions) Alexa fluor 488 and 568 secondary antibodies, by Thermo Fisher Scientific (1 mentions) Slowfade, by Thermo Fisher Scientific (1 mentions) Dapi nuclear staining, by Merck Group (1 mentions) C kit, by R&D Systems (1 mentions) 1140 autocut microtome, by Reichert Technologies (1 mentions)

11 protocols using α sarcomeric actin

1

Apoptosis Detection in Cardiac Tissue

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Apoptosis detection was performed on 10 µm frozen sections with the ApopTag Peroxidase In Situ Oligo Ligation Kit (Millipore, Germany) according to the manufacture instructions. To evaluate apoptosis rate in cardiomyocytes and non-cardiomyocytes, immunostaining for cardiomyocyte marker α-sarcomeric actin (Sigma-Aldrich, Germany) was performed.
Petersen J., Kazakov A., Böhm M., Schäfers H.J., Laufs U, & Abdul-Khaliq H. (2018). Cardiopulmonary bypass reduces myocardial oxidative stress, inflammation and increases c-kit+CD45− cell population in newborns. Journal of Translational Medicine, 16, 111.
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2

Cardiac Immune Cell Profiling Post-Myocardial Infarction

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Mice (N = 10–12 per group) were sacrificed 30 days after AMI.
Following echocardiography, hearts were harvested as previously described [16 (link)]. Hearts were stained against: Rabbit
anti-mouse IBA1 (Novus Biologicals, CO) overnight at 4°C. After washing,
sections were incubated with secondary antibody conjugated to Alexa Fluor 488
(1:500, Invitrogen, Carlsbad, CA), then incubated with Sudan Black B (Sigma
Aldrich, St. Louis, MO) for 30 minutes and subsequently incubated with DAPI
nuclear counterstain. A similar protocol was used to prepare heart sections from
mice on baseline, day 7 and day 30 for capillary density assessment using FITC-
Isolectin B4 (FL1201, Vector Labs, Burlingame, CA). Apoptotic cells in heart
sections were identified at day 1 by terminal deoxynucleotidyl
transferase-mediated dUTP nick end-labeling (TUNEL) staining using the In-Situ
Cell Death Detection Kit and α-sarcomeric actin (1:200 dilution, Sigma
Aldrich, St. Louis, MO) according to the manufacturer’s protocol. DAPI
counterstaining was followed by a final PBS wash.10–15 adjacent
pre-infarct and remote zones per section were analyzed (1–2
sections/animal) at 40x magnification using Nikon Confocal Microscope A1 in the
University of Kentucky Confocal Microscopy facility.
Tripathi H., Al-Darraji A., Abo-Aly M., Peng H., Shokri E., Chelvarajan L., Donahue R., Levitan B.M., Gao E., Hernandez G., Morris A.J., Smyth S.S, & Abdel-Latif A. (2020). Autotaxin Inhibition Reduces Cardiac Inflammation and Mitigates Adverse Cardiac Remodeling After Myocardial Infarction. Journal of molecular and cellular cardiology, 149, 95-114.
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3

Cardiac Tissue Analysis Post-Myocardial Infarction

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After final echocardiography and peripheral blood collection, hearts were harvested in diastole with saturated KCl and CdCl (100 mM) injected through the apex into the LV cavity. The LV apex was then cannulated and the heart perfused with PBS followed by 10% buffered formalin at 75 mmHg with severed inferior vena cava serving as the outlet. Hearts were then cut into 2 mm cross sectional slices and processed for paraffin embedding. Slices were cut into 4 μm sections for histology and immunofluorescent staining. LV area, LV cavity area, and infarct area were measured in Masson’s Trichrome stained sections. Images were acquired digitally and areas were measured using NIH ImageJ (v1.37). Cell turn-over was evaluated by staining against BrdU (Roche, Indianapolis, IN) and α-sarcomeric actin (Sigma, St Louis, MO) or myosin heavy chain (MHC) (Abcam, Cambridge, MA) and counting BrdUposα-SApos/MHCpos cells in both ischemic and remote regions. GFP bone marrow cell retention was assessed by anti-GFP staining in infarct border regions (Abcam). Capillary density was measured by staining against FITC- isolectin B4 (FL1201, Vector Labs, Burlingame, CA) in cardiac sections. 10–15 adjacent areas in the peri-infarct and remote zones per section were analyzed (1–2 sections/animal) at 40x magnification using Nikon Confocal Microscope A1 in the University of Kentucky Confocal Microscopy facility.
Klyachkin Y.M., Idris A., Rodell C.B., Tripathi H., Ye S., Nagareddy P., Asfour A., Gao E., Annabathula R., Ratajczak M., Burdick J.A, & Abdel-Latif A. (2018). Cathelicidin Related Antimicrobial Peptide (CRAMP) Enhances Bone Marrow Cell Retention and Attenuates Cardiac Dysfunction in a Mouse Model of Myocardial Infarction. Stem cell reviews, 14(5), 702-714.
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4

Isolation of Mouse Ventricular Cardiomyocytes

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Primary cultures of mouse ventricular cardiomyocytes were prepared from hearts of 1 to 2-day-old neonatal C57BL6/J mice (Jackson, Bar Harbor, ME) as described previously [1] (link). In brief, the cells were isolated at 37°C for 8 min with 0.1% trypsin in Hanks' balanced salt solution (HBSS) without Ca2+ and Mg2+ (pH 7.4). The first cell suspension was discarded, whereas the subsequent suspensions were added to trypsin inhibitor solution in cold HBSS with Ca2+ and Mg2+ (pH 7.4) until all cardiac cells were isolated (5 to 6 cycles). To remove fibroblasts, the isolated cells were preplated for 90 min at 37°C. The resulting supernatants were then centrifuged and plated at a density of 0.6×106 on laminin-coated coverslips with MEM supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 50 U/ml penicillin, and 1.5 µM vitamin B12 (Sigma, St. Louis, MO). Myocyte purity was ∼90% as determined by immunofluorescent staining for α-sarcomeric actin and myosin heavy chain (Sigma, St. Louis, MO). Experiments were performed on 6–8 day cultures.
Zhu X., Shao Z.H., Li C., Li J., Zhong Q., Learoyd J., Meliton A., Meliton L., Leff A.R, & Vanden Hoek T.L. (2014). TAT-Protein Blockade during Ischemia/Reperfusion Reveals Critical Role for p85 PI3K-PTEN Interaction in Cardiomyocyte Injury. PLoS ONE, 9(4), e95622.
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5

Cardiac Tissue Immunohistochemistry and Collagen Staining

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Hearts were fixed in 10% formalin for 24 h, followed by an overnight dehydration in 70% ethanol. Hearts were sectioned into apex, mid and base, and processed for paraffin embedding. 10 µm thick slices of heart from the apex, mid and base regions were cut using a Reichert-Jung, 1140/autocut microtome and slide mounted for direct immunohistochemical fluorescent staining. Sections were deparaffinised with Histoclear, prior to rehydration and antigen retrieval (boiling in citrate buffer for 30 min). Immunofluorescent antibody labelling (c-kit, R&D; α-sarcomeric actin, Sigma; AlexaFluor-488 and -568 secondary antibodies, Thermo Fisher) proceeded according to standard methods, with DAPI nuclear staining (Sigma) and coverslip mounting with Slowfade (Life Technologies). For collagen staining, slides were rinsed with Histoclear twice, prior to rehydration and staining with Sirius Red for 60 min, followed by 0.01 M HCl wash to remove excess stain. The slides were dehydrated and mounted in DPX prior to viewing. Slides were viewed using a Zeiss LSM 700 confocal microscope, and image analysis (cell counts or area calculations) performed using ImageJ, with 18 random fields of view analysed per subject (six individual subjects per group).
Smith A.J., Ruchaya P., Walmsley R., Wright K.E., Lewis-McDougall F.C., Bond J, & Ellison-Hughes G.M. (2022). Receptor tyrosine kinase inhibitors negatively impact on pro-reparative characteristics of human cardiac progenitor cells. Scientific Reports, 12, 10132.
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6

miR-34a Detection in Cardiac Tissue

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MiR-34a was detected by FISH according to protocol previously described [54 (link)] and modified as follow. Tissues sections were digested by 0.5μg/ml proteinase-K for 10′ at RT. miRCURY LNA detection probe hsa-miR-34a, 5′-DIG and 3′-DIG labeled and control probe LNA U6 snRNA, 5′-DIG labeled (Exiqon) were used at 50 nM and 10 nM respectively and denatured as suggested by manufacturer's instructions. To detect signal Anti-Digoxigenin antibody (Abcam) and TSA Fluorescein Plus evaluation kit (Perkin Elmer) were used. rCPCs were identified by c-kit (Santa Cruz Biotechnology), myocytes were labeled with α-sarcomeric actin (Sigma-Aldrich) and nuclei were stained with DAPI (Sigma-Aldrich). Samples were analyzed with a Zeiss LSM700 confocal microscope.
Piegari E., Russo R., Cappetta D., Esposito G., Urbanek K., Dell'Aversana C., Altucci L., Berrino L., Rossi F, & De Angelis A. (2016). MicroRNA-34a regulates doxorubicin-induced cardiotoxicity in rat. Oncotarget, 7(38), 62312-62326.
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7

Cardiac Protein Extraction and Analysis

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Freshly isolated mouse ventricles were pulverized in liquid nitrogen by mortar and pestle. Protein lysates were harvested in radioimmunoprecipitation (RIPA) buffer (20 mM Tris-HCl; pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate) supplemented with protease (Roche, 11836170001) and phosphatase (Pierce, 184724) inhibitors. Protein quantification was carried out by Bradford assays (Sigma, B6916). Standard protein electrophoresis and transfer were performed16 (link). Antibodies and their dilution are listed below. Tmem65 antibody (1:1000 dilution; Sigma, HPA025020), Connexin43 antibody (1:1000; Sigma, C6219), α-Sarcomeric Actinin (1:1,000; Sigma, A7811), α-Sarcomeric Actin (1:1,000; Sigma, A2172), MyHC (1:40; DSHB, MF20), β-Actin (1:200; SC-47778, Santa Cruz Biotechology), cTnT (1:40; DSHB, CT3), MYH7 (1:40, DSHB, A4.840), Tubulin (1:40; DSHB, E7), FHL1 (1:1000; ab49241), Erk1/2 (1:1,000; Cell Signaling Technology, 9102), Phospho-Erk1/2 (1:1,000; Cell Signaling Technology, 9101), β1 (1:1,000, Cell Signaling Technology), NaV1.5 (1:1000; ASC-005, Alomone Labs), N-Cadherin (1:30 unconcentrated hybridoma supernatant; MNCD2, DSHB), and FLAG (1:1,000; Sigma, F1804).
Teng A.C., Gu L., Di Paola M., Lakin R., Williams Z.J., Au A., Chen W., Callaghan N.I., Zadeh F.H., Zhou Y.Q., Fatah M., Chatterjee D., Jourdan L.J., Liu J., Simmons C.A., Kislinger T., Yip C.M., Backx P.H., Gourdie R.G., Hamilton R.M, & Gramolini A.O. (2022). Tmem65 is critical for the structure and function of the intercalated discs in mouse hearts. Nature Communications, 13, 6166.
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8

Immunofluorescence Assay for Protein Detection

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Immunofluorescence was performed as previously described (Briggs et al., 2013 (link)) with antibodies recognizing the following antigens: Smo (Abcam AB72130); Lef1 (Cell Signaling 22305); cyclin D1 (Cell Signaling 29785); Islet1 (Isl1; DSHB 39.4D5), myosin heavy chain (MF20; DSHB), pSMAD1,5,8 (pSMAD1/5/8; Millipore; catalogue#: AB3848), α-sarcomeric actin (sAct; Sigma; catalogue#: A2172), FilaminA (FLNA; Epitomics; catalogue#: 2242-1), pSMAD3 (pSMAD3; Epitomics; catalogue#: 1880-1), Crtl1 (DSHB; 9/30/8-A-4); HABP (Seikagaku; catnr 400763); Ki67 (Dako; catnr M7248), beta-catenin (Cell Signaling; 95815), Axin2 (Abcam; ab 32197), and EGFP (Abcam; ab 13970). Secondary antibodies (Jackson Immunoresearch) included anti-rabbit TRITC (711-025-152), anti-rabbit FITC (711-095-152), anti-rabbit Cy5 (711-175-152), anti-mouse TRITC, anti-mouse FITC (715-095-151), anti-mouse Cy5 (715-175-150), anti-rat TRITC (715-025-150), anti-rat FITC (715-095-150), anti-rat Cy5 (711-175-150), and anti-chicken Cy5 (703-495-155). The ApopTag fluorescein direct in situ kit (Millipore; catalogue#: S7160) was used to label apoptotic cells. Nuclei were visualized using DAPI (4′,6-diamidine-2-phenylidole-dihydrochloride, Invitrogen; Slowfade Gold Antifade Reagent with DAPI; catalogue#: S36938) and fluorescence visualized using a Zeiss AxioImager II microscope.
Briggs L.E., Burns T.A., Lockhart M.M., Phelps A.L., Van den Hoff M.J, & Wessels A. (2015). Wnt/β-Catenin and Sonic Hedgehog Pathways Interact in the Regulation of the Development of the Dorsal Mesenchymal Protrusion. Developmental dynamics : an official publication of the American Association of Anatomists, 245(2), 103-113.
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9

Histological Analysis of Cardiac Tissue

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Histological studies were performed in formalin‐fixed, paraffin‐embedded, 4‐μm‐thick heart sections. The procedures for Masson's trichrome, picrosirius red, and immunohistochemistry staining and analyses have been described.3, 7, 8, 9, 11, 12 Immunohistochemistry staining was performed with specific antibodies against CD45, wheat germ agglutinin, α‐sarcomeric actin, BrdU , and IdU (Sigma). To assess the fate of the transplanted male CPCs, Y chromosome was detected by fluorescence in situ hybridization according to the modified manufacturer's protocol (ID Labs, London, ON).3, 8, 13
Tang X., Nakamura S., Li Q., Wysoczynski M., Gumpert A.M., Wu W., Hunt G., Stowers H., Ou Q, & Bolli R. (2018). Repeated Administrations of Cardiac Progenitor Cells Are Superior to a Single Administration of an Equivalent Cumulative Dose. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 7(4), e007400.
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10

Cardiac Protein Fractionation and Immunodetection

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Triton x-100 soluble and insoluble protein lysates of mouse ventricles were prepared as previously described11 (link). The following antibodies were used: phospho-mTORser2448, total mTOR, phospho-p70S6 kinaseThr389, total p70S6 kinase, phospho-4E-BPThr37/46, total 4E-BP1, phospho-AMPKThr172, total AMPK, methyl-arginine, (Cell Signaling; Beverly, MA), ADK, (Santa Cruz Biotechnology; Santa Cruz, CA), atrial natrurietic peptide (Peninsula biolabs; San Carlos, CA), Desmin (Abcam; Cambridge, MA), and detyrosinated tubulin (Abcam; Cambridge, MA and Upstate Biotechnology; Lake Placid, NY), β-MHC, α-sarcomeric actin, β-actin (Sigma; St Louis, MO).
Fassett J., Xu X., Kwak D., Zhu G., Fassett E.K., Zhang P., Wang H., Mayer B., Bache R.J, & Chen Y. (2019). Adenosine Kinase attenuates cardiomyocyte microtubule stabilization and protects against pressure overload-induced hypertrophy and LV dysfunction. Journal of molecular and cellular cardiology, 130, 49-58.
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