Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

Summary

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Here, we describe a protocol to evaluate sarcoplasmic reticulum Ca²⁺ reserve and diastolic calcium removal function in cardiomyocytes by a calcium imaging system.

Abstract

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca²⁺ reservoir for contraction, which reuptakes intracellular Ca²⁺ during relaxation. The SR Ca²⁺ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca²⁺ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca²⁺ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa²⁺ reserve and diastolic Ca²⁺ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca²⁺ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca²⁺ release, we preset an automatic perfusion switch program by interlinking the stimulation system and the perfusion system. Then, the mono-exponential curve fitting was used for analyzing decay time constants of calcium transients and caffeine-induced calcium pulses. Accordingly, the contribution of the SR Ca²⁺-ATPase (SERCA) and Na⁺-Ca²⁺ exchanger (NCX) to diastolic calcium removal was evaluated.

Introduction

Intracellular calcium ([Ca²⁺]_i) recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes¹. As we know, the calcium-induced Ca²⁺ release initiates the excitation-contraction coupling, which translate the electrical signal to contraction. Membrane depolarization activates the sarcolemmal L-type Ca²⁺ channels, which induce Ca²⁺ release from SR into the cytoplasm via ryanodine receptors 2 (RyR2). The transient elevated cytoplasmic Ca²⁺ initiates contraction of myofibrils. During the diastole, cytoplasmic Ca²⁺ is reuptaken into the ....

Protocol

All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee at Experimental Research Center, China Academy of Chinese Medical Sciences and Zhejiang University.

1. Solution Preparation

1. Prepare all solutions as described in Table 1.

2. Isolation of Left Ventricular (LV) Cardiomyocytes

NOTE: LV cardiomyocytes are isolated enzymatically using a Langendroff perfusion system as previously described^7,8,9

Results

              Here, we illustrate streptozotocin (STZ)-induced diabetic rats (DM) and age-matched Sprague-Dawley (SD) rats for example. 8-week-old male SD rats (200 ± 20 g) received a single intraperitoneal injection of STZ (70 mg/kg, ip) for the DM group or citrate buffer for the control group. One week after STZ administration, rats with blood glucose > 16.7 mmol/L were considered diabetic. After 8 weeks, the LV myocytes were enzymatically isolated. After calcium reintroduction, ther.......

Discussion

Calcium flux released from the SR is the major Ca²⁺ source for systole in the heart. To some extent, the amplitude of SR Ca²⁺ content and the fractional Ca²⁺ released from the SR reflect the SR Ca²⁺ reserve available for cardiac contraction. On the other hand, the Ca²⁺ reserve of SR depends on the ability of SR Ca²⁺ reuptake, Ca²⁺ leak of SR, and their balance across the SR during diastole^12,1.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NaCl	Alfa Aesar	012314
MgCl2	Alfa Aesar	012315
KCl	Alfa Aesar	11595
HEPEs	Sigma	H3375
D-Glucose	Alfa Aesar	A16828
NaOH	Alfa Aesar	A18395
KOH	Alfa Aesar	A18854
KH2PO4	Alfa Aesar	011594
MgSO4	Alfa Aesar	3337
L-Glutamic	Alfa Aesar	A15031
Taurine	Alfa Aesar	A12403
EGTA	Sigma	E3889
Collagenase A	Roche	10103586001
Collagenase Type II	Worthington	LS004177
BSA	Roche	735094
caffeine	Sigma	C0750
Fura-2 AM	Invitrogen	F1201
Microscope	Olympus	Olympus IX 71
Langendorff system	Beijing Syutime Technology Co	PlexiThermo-S-LANGC
Micromanipulator	Marchauser	MM33 links
Perfusion chamber	IonOptix	FHD
Valve Controlled Gravity Perfusion System	ALA	VC 3-8
valve commander software	ALA	VC 3 1.0.1.2
Precision flow regulator	Delta Med	3204315
Multi-Barrel Manifold Perfusion Pencil	AutoMate Scientific	04-08-[360]
Micron Removable Tip	AutoMate Scientific	360um i.d.
Fluorescence Measurement and Cell Dimensioning Systems	IonOptix	Hyperswitch
Recording software	IonOptix	IonWizard 6.2.59
Stimulator	IonOptix	MyoPacer EP
Sprague-dawley rats	Beijing Vital River Laboratory Animal Technology Co.	SCXK 2016-01-007436