title: A simplified protocol for culture of murine neonatal cardiomyocytes on nanoscale keratin coated surfaces.
abstract: OBJECTIVE
  We aim to develop a simple, efficient and cost-effective protocol for culturing the neonatal cardiomyocytes using keratin derived from human hair, which can be used for studying cardiac hypertrophy in vitro.
  
  
  METHODS
  Keratin was extracted from human hair and applied as nanoscale coating onto the culture dishes. Physical parameters such as surface morphology and roughness of the coating were studied by SEM and AFM. Cardiomyocyte specific markers were assessed by immunofluorescence. Signaling pathways activated in hypertrophy were analyzed by western blotting and changes in the expression of fetal genes were analyzed by qPCR. The changes in the calcium fluxes were observed microscopically using Fluo-4.
  
  
  RESULTS
  Keratin coated surfaces displayed a uniform coating and comparable roughness across dishes. Our optimized protocol for isolating cardiomyocytes yielded up to ~106cells per heart. Characterization of cardiomyocytes with specific markers revealed that they can attach, grow and show spontaneous contractions on keratin-coated substrates similar to fibronectin-coated surfaces. Phenylephrine (PE) treated cardiomyocytes grown on keratin-coated substrates exhibited increased cell size, sarcomere organization and perinuclear ANP expression indicating the development of cardiac hypertrophy. In addition, we observed increased activation of Akt and ERK pathways, induction of the fetal genes and increased protein synthesis upon PE treatment, which are characteristics of cardiomyocyte hypertrophy. The protocol was extended to mouse cardiomyocytes and found to show similar results upon examination.
  
  
  CONCLUSIONS
  We demonstrate that keratin can act as an efficient yet cost effective alternative substrate for the attachment, growth and differentiation of neonatal murine cardiomyocytes.
authors.affiliation: Centre for Biosystems Science and Engineering, Indian Institute of Science, Bengaluru 560012, India.
authors.forename: Aditi
authors.initials: A
authors.lastname: Jain
authors.affiliation: Cardiovascular and Muscle Research Laboratory, Department of Microbiology and Cell Biology, Indian Institute of Science, Bengaluru- 560012, India.
authors.forename: Venkatraman
authors.initials: V
authors.lastname: Ravi
authors.affiliation: Cardiovascular and Muscle Research Laboratory, Department of Microbiology and Cell Biology, Indian Institute of Science, Bengaluru- 560012, India.
authors.forename: Jaseer
authors.initials: J
authors.lastname: Muhamed
authors.affiliation: Centre for Biosystems Science and Engineering, Indian Institute of Science, Bengaluru 560012, India; Department of Materials Engineering, Indian Institute of Science, Bengaluru- 560012, India. Electronic address: kchatterjee@materials.iisc.ernet.in.
authors.forename: Kaushik
authors.initials: K
authors.lastname: Chatterjee
authors.affiliation: Centre for Biosystems Science and Engineering, Indian Institute of Science, Bengaluru 560012, India; Cardiovascular and Muscle Research Laboratory, Department of Microbiology and Cell Biology, Indian Institute of Science, Bengaluru- 560012, India. Electronic address: rsundaresan@mcbl.iisc.ernet.in.
authors.forename: Nagalingam R
authors.initials: NR
authors.lastname: Sundaresan
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D009320:Atrial Natriuretic Factor / Q000096:biosynthesis / Q000235:genetics; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D006332:Cardiomegaly / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology*; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004195:Disease Models, Animal; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D053564:Keratins, Hair-Specific / Q000494:pharmacology*; D020935:MAP Kinase Signaling System; D051379:Mice; D008807:Mice, Inbred BALB C; D018625:Microscopy, Atomic Force; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D053758:Nanoparticles; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D015398:Signal Transduction
chemical_list: D017077:Culture Media, Conditioned; D053564:Keratins, Hair-Specific; D009320:Atrial Natriuretic Factor; D004247:DNA; D002118:Calcium
keywords: Cardiac hypertrophy; Cardiomyocytes; Human hair; Keratin; Nanoscale coating