Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Summary

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

Abstract

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis—in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before—and provide some representative results.

Introduction

The heart responds to the injury of ischemia (I) and reperfusion (R) in a dynamic fashion. However, there is little insight into acute changes in protein synthesis during the response. To address this, we took advantage of the well-established method of polysome profiling¹ to identify changes in protein abundance that reflect redistribution of ribosomes and translational regulatory factors from cytosol to polysomes, and the increase in newly synthesized proteins (NSPs). In the setting of I/R, the increase in new protein synthesis occurs in a time frame that is inconsistent with transcription of new mRNAs²; moreover, disc....

Protocol

All animal studies were performed in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee of Cedars-Sinai Medical Center.

1. Langendorff Perfusion of Mouse Heart

1. Langendorff perfusion of mouse heart with ischemia and reperfusion
   1. Administer intraperitoneal pentobarbital sodium 70 mg/kg to the adult mouse (8-week-old, male, C57BL6/j). Confirm deep anesthesia by lack of withdrawal to toe pinch.
   2. Anticoagulate with intraperitoneal heparin 500 U/kg.
   3. Open the chest via sternal incision. Open the diaphragm and cut along the costal bor....

Results

mRNA analysis
mRNA results can be expressed as a distribution of a particular mRNA in each fraction (Figure 3A); for quantification, combine polyribosomal translating fractions and compare to the non-translating fraction (Figure 3B), presenting a ratio of mRNA abundance in translating to nontranslating fractions. Additional information is gained by examining the high efficiency polysome fractions separately from low-.......

Discussion

Polysome profile analysis allows for the study of protein translation by analyzing the translational state of a specific mRNA or the whole transcriptome^6,7. It is also of great help when local translation needs to be studied such as synaptosomes⁸. Traditionally, this method involves the separation of mono- and polyribosomes and the associated mRNAs on a sucrose gradient which could be coupled with genomic or proteomic techniques to obtain .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Pentobarbital	Vortech Phamaceuticals	9373	for euthansia
Heparin	Sagent	103424	used in langendorff preparation
forceps	Fine Science Tools	91110-10	used to hang the heart
Langendorff system	Radnoti + home made	n/a	A 'four heart' system consisting of custom blown glass, tubing and water baths
NaCl	Sigma	S7653-5KG	Krebs buffer and Sucrose gradient
KCl	Sigma	P5405	Krebs buffer and Lysis buffer
KH2PO42	Sigma	P-5504	Krebs buffer
MgSO4	Sigma	M7774-500G	Krebs buffer
Glucose	Sigma	G5767	Krebs buffer
CaCl2	Sigma	C1016-500G	Krebs buffer
Sucrose powder	Sigma	S0389-1KG	Sucrose gradient
MgCl2	Sigma	208337	Sucrose gradient and Lysis buffer
Tris-base	Sigma	T1503-1KG	Sucrose gradient and Lysis buffer
Xylene Cyanole	Sigma	X-4126	Sucrose gradient
Cycloheximide	Sigma-aldrich	239763	Sucrose gradient and Lysis buffer
RNaseOUT	Life Technologies	C00019	RNAse inhibitor for Lysis buffer
Igepal CA-360 (NP40)	Sigma	I3021	Lysis buffer
Protease Inhibitor Cocktail tablets, EDTA free	Roche	5056489001
Tube, Thinwall, Ultra-Clear, 13.2 mL, 14 mm x 89 mm	Beckman Coulter	344059
Ultracentrifuge	Beckman	LE-80K	Ultracentrifugation of the gradients
Rotor	Beckman	SW41	Ultracentrifugation of the gradients
Biologic LP (pump)	Biorad	731-8300	Fractionation of the gradients
BioFrac	Biorad	741-0002	Fractionation of the gradients
Eppendorf RNA/DNA LoBind microcentrifuge tubes, 2 mL tube	Sigma	Z666513-100EA	Gradient fraction and RNA extraction
TRIzol Reagent	Life technologies	AM9738	RNA extraction
Luciferase Control RNA	Promega	L4561	RNA extraction
Chloroform	Fisher Scientific	C606-4	RNA extraction
Glycogen, RNA grade	Thermo Fisher Scientific	R0551	RNA extraction
Isopropanol	Sigma	I9516	RNA extraction
Ethanol	Sigma	E7023-1L	RNA extraction
iScript cDNA Synthesis Kit	BioRad	170-8891	Reverse transcription
iTaq Universal SYBR Green Supermix	BioRad	175-5122	Quantative PCR
miRNeasy Micro Kit (50)	Qiagen	217084	Kit for total RNA isolation
miScript II RT Kit (50)	Qiagen	218161	Kit for miRNA reverse transcription
miScript Sybr Green PCR Kit (200)	Qiagen	218073	Kit for real-time PCR expression analysis of miRNAs
Centrifuge 5424R	Eppendorf		For centrifugation of 1.5 mL or 2.0 mL tubes at different temperatures. Max speed - 21130 x g
Centrifuge 5810R	Eppendorf		For real-time PCR plate centrifugation at different temperatures. Max speed - 2039 x g
My Cycler Thermal Cycler	Bio-Rad		For reverse transcription
CFX96 Real-Time System/C1000 Touch Thermal Cycler	Bio-Rad		For real-time PCR analysis
miRNeasy Serum/Plasma Spike-in Control	Qiagen	219610	For quality control of RNA isolation
Hard-Shell 96-Well PCR Plates, low profile, thin wall, skirted, green/clear	Bio-Rad	HSP9641	For real-time PCR analysis
Microseal 'B' PCR Plate Sealing Film, adhesive, optical	Bio-Rad	MSB1001	For real-time PCR plate sealing
Research plus Single-Channel Pipette, Gray; 0.5-10 µL	Eppendorf	UX-24505-02	For pipetting
PIPETMAN Classic Pipets, P20	Gilson	F123600G	For pipetting
PIPETMAN Classic Pipets, P200	Gilson	F144565	For pipetting
Rainin L-1000XLS Pipet-Lite XLS LTS Pipette 100-1000 µL	Gilson	17011782	For pipetting
Glycogen, RNA grade	Thermo Fisher Scientific	R0551	Improves total RNA isolation efficiency
Posi-Click 1.7 mL Tubes, natural color	Denville	C2170	RNA isolation and storage; reagent mix
Thermal Cycling Tubes -0.2 mL Individual Caps, Standard 0.2 mL tubes with optically	Denville	C18098-4 (1000910)	Reverse transcription reaction
Sharp 10 Precision barrier Tips	Denville	P1096-FR	For pipetting
Sharp 20 Precision barrier Tips	Denville	P1121	For pipetting
Sharp 200 Precision barrier Tips	Denville	P1122	For pipetting
Tips LTS 1 mL Filter	Rainin	RT-L1000F	For pipetting
miScript Primer Assay (200)	Qiagen	(it changes according to the miRNA)	For real-time PCR analysis
Gradient Master ver 5.3 Model 108	BioComp Instruments		For preparation of sucrose gradients
trichloroacetic acid	Sigma Aldrich	T6399
acetone	Sigma Aldrich	650501
Tris hydrochloride	Amresco	M108
dithiothreitol	Fisher Scientific	BP172
iodoacetamide	Gbiosciences	RC-150
sequencing grade modified trypsine, porcine	Promega	V5111
ammonium bicarbonate	BDH	BDH9206
formic acid, Optima LC/MS	Fisher Chemical	A117
methanol, Optima LC/MS	Fisher Chemical	A454
acetonitrile, Optima LC/MS	Fisher Chemical	A996
Protein LoBind tubes 0.5 mL	Eppendorf AG	22431064
Protein LoBind tubes 1.5 mL	Eppendorf AG	22431081
HLB µElution plate 30 µm	Oasis	186001828BA
SpeedVac concentrator	Thermo Scientific	Savant SPD2010
sonicator	Qsonica	Oasis180
centrifuge	Thermo Scientific	Sorvall Legend micro 21R
LC trap column PepMap 100 C18	Thermo Scientific	160454
LC separation column PepMap RSLC C18	Thermo Scientific	164536
mass spectrometer	Thermo Scientific	Orbitrap Elite ion trap mass spectrometer
MSConvert software	ProteoWizard Toolkit
Sorcerer-SEQUEST software	Sage-N Research, Inc.