Investigation of Phospholipase C-

1
Investigation of Phospholipase C-ß1b Splice
Variants on Function in H9c2 Cells

• Chelsea Parker
• Dr. Theresa Filtz
• Oregon State University
• HHMI Program
• Summer 2009

2
Importance

• Hypertrophy
• Congestive Heart Failure
• Causes obesity, birth defects, high blood
  pressure
• Widespread over 79 million
• people affected
• Treatments Digitalis,
• Beta blockers, diuretics

http//odyb.net/physical-health/9-types-of-disease
s-caused-by-obesity/
3
Background

• Phospholipase C-ßs are stress activated enzymes
  present in many tissues and organs
• Only the tail structure of PLC-ß1b differs from
  other phospholipase C-ß subtypes

http//atlasgeneticsoncology.org/Genes/PLCB1ID4174
2ch20p12.html
4
Molecular Pathway

• PLC-ß1b in heart cells
• Signaling

http//journals.prous.com/journals/dof/19992407/ht
ml/df240759/images/Yerxa2.gif
5
Previous Research

2.0
Cell area
1.0
0.0
No virus
PLCb1a
PLCb1b
6
Project Description

• Study H9c2 cells
• Induce hypertrophy
• Assay changes in native PLC-ß1 levels
• Transfect H9c2 cells with both PLC-ß1 variants
• Assay for hypertrophy
• Create mutants of PLC-ß1b
• Over-express the PLC-ß1b mutants in H9c2 cells
• Assay for hypertrophy

7
Hypothesis

• The tail section (extreme C-terminus) of PLC-ß1b
  is responsible for PLC-ß1b -induced heart cell
  enlargement
• Prediction
• Over-expression of PLC-ß1b tail segments will
  lead to decreased heart cell enlargement in H9c2
  cells

8
Results

• H9c2 cell maintenance techniques
• Identification of PLCB-1 in H9c2 cells
• Hypertrophy
• Transformation of GFP,
• PLC-ß1a, and PLC-ß3
• Transfection and expression of GFP in H9c2
• cells

7/14/2009
9
Continued Research

• Optimize transfection efficiency
• Generate a PLC-ß1b construct with PCDNA as the
  vector
• Create PLC-ß1b mutants
• Transfect H9c2 cells with PLC-ß DNA and PLC-ß1b
  mutant DNA

10
Special Thanks To

• Dr. Theresa Filtz
• Dr. Satin Salehi
• Dr. Kevin Ahern
• Howard Hughes Medical Institute
• Ray, Frances, and Dale Cripps Scholarship Fund