Measuring Ca2+-dependent Ca2+-uptake activity in the mouse heart.

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Measuring Ca2+-Dependent Ca2+-Uptake Activity in the Mouse Heart Tine Holemans, Ilse Vandecaetsbeek, Frank Wuytack and Peter Vangheluwe Cold Spring Harb Protoc; doi: 10.1101/pdb.prot076893 Email Alerting Service Subject Categories

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Protocol

Measuring Ca2+-Dependent Ca2+-Uptake Activity in the Mouse Heart Tine Holemans, Ilse Vandecaetsbeek, Frank Wuytack, and Peter Vangheluwe1 Laboratory of Cellular Transport Systems, Department of Cellular and Molecular Medicine, KU Leuven, B3000 Leuven, Belgium

The apparent Ca2+ affinity of the isoforms of the sarco/endoplasmic reticulum Ca2+ ATPase SERCA2 is controlled primarily by two proteins, phospholamban (PLB) and sarcolipin (SLN). The rate of ATPdriven Ca2+ uptake into sarcoplasmic reticulum (SR)-derived vesicles can be monitored by a technique in which the net uptake of 45Ca2+ in the form of an intravesicular calcium oxalate precipitate is recorded. Here, we present details of a modification of such a protocol for determining the apparent Ca2+ affinity of the Ca2+ pump, and its control by various regulators, in crude homogenates of mouse heart.

MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this article for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.

Reagents

Adenosine triphosphate (Na-ATP; pH 7.0) Antibodies Bovine serum albumin (BSA) Bicinchoninic acid (BCA) [45Ca2+]CaCl2 (range 1–20 mCi/mL) CaCl2 (0.1 M; analytical grade, commercially available) EGTA (ethylene glycol tetraacetic acid) Heparin Homogenization buffer for Ca2+-uptake assay Liquid nitrogen Master mix for Ca2+-uptake assay MnCl2 (1 mM) MilliQ (mQ) deionized water (Millipore) PBS (phosphate-buffered saline), prechilled on ice λ-protein phosphatase (λ-PPase) 1

Correspondence: [email protected]

© 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot076893

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Measuring Ca 2+ -Dependent Ca 2+ Uptake in Heart

Scintillation liquid Sodium pentobarbital Software for calculating kinetic parameters (Origin, MicroCal Software) Thapsigargin (100 nM) Wash buffer for Ca2+-uptake assay

Equipment

Cryotubes (2 mL) Dissection tools (standard scissors and forceps suitable for mouse dissections) Eppendorf tubes with cap (2 mL) Falcon tubes (15 mL and 50 mL) Filtration unit connected to a vacuum pump with a collector for radioactive waste Gauze swab (5 × 5 cm2) Microcentrifuge (coolable) Millipore filters (HAWP filters, 0.45 µm pore size) Protein electrophoresis module (4%–12% Bis/Tris gels and SDS–PAGE running buffer) Protein transfer module and transfer buffer for standard western blotting PVDF membrane (0.22 µm, Immobilon) Sarstedt tubes (11.5 mL, polypropylene) Scintillation vials, screw cap Scintillation counter Spectrophotometer (562 nm) Potter homogenizer (Teflon/glass, 5 cm3), preferably motorized Toothpick (or needle) Transparent tubes (Sarstedt Röhren 11.5 mL tubes) Ultra-Turrax homogenizer (20,000 rpm) using a flat-bottomed hard-tissue-homogenizing probe (diameter 8–12 mm) Vortex Water bath (37˚C)

METHOD Isolation of Mouse Ventricles

1. Inject mice (age 12–16 wk) intraperitoneally with 100 µL heparin and 5 min later anaesthetize them by intraperitoneal injection of sodium pentobarbital according to body mass (50 mg/kg). Handle the mice as consistently as possible and avoid stressing them. Follow your local animal-welfare procedures.

2. Open the chest with the dissection tools as quickly as possible, rapidly excise the whole heart, and transfer it into 50 mL of ice-cold PBS. Dissection should take 60 min) (Ji et al. 1999). Storing on ice for > 1 h is not recommended as this leads to significant loss of SERCA2a activity (Ji et al. 1999). To average out the loss of activity over time, measure the first pCa series from low to high [Ca2+] and the second series from high to low [Ca2+]. Importantly, protease and phosphatase inhibitors should be freshly added to the homogenization buffer to, respectively, prevent protein digestion of the tissue homogenates and to maintain the endogenous phosphorylation level of PLB. This will greatly enhance the stability of the homogenate. Problem (Step 29): Not all the Ca2+ uptake is related to the activity of SERCA2a. Solution: Add 100 nM thapsigargin (Ji et al. 1999), a highly specific SERCA inhibitor, to the pCa-

reaction-mix to determine how much Ca2+ uptake is related to the SERCA pump. The IC50 of thapsigargin is subnanomolar (Vangheluwe et al. 2009). Problem (Step 29): The Vmax values are low or differ from the ones reported in the literature. Solution: The Vmax depends somewhat on the method of choice for determining the protein con-

centration, but generally falls within the range of 20–100 nmol Ca2+/mg/min (Ji et al. 1999; Zhao et al. 2003; Vangheluwe et al. 2006b). Uptake values that are too low can point to poor quality of the homogenate, leaky SR vesicles, low-quality ATP, an actual [Ca2+]free that is too low owing to excess buffering or to open ryanodine receptor channels, leading to Ca2+ release. Ensure that the homogenization procedure is not too harsh and that all solutions are well prepared.

Problem (Step 29): The Km values differ from those reported in the literature. Solution: Various laboratories report different Km values, ranging for wild-type mice from 0.2–0.5 µM

(Ji et al. 1999; Zhao et al. 2003; Vangheluwe et al. 2006b). This might reflect differences in pH, temperature or actual free-Ca2+ concentrations in the assay. Note that the calculations of the freeCa2+ concentrations depend on the parameters or on the software program, which might use different equilibrium constants. Our method renders values for wild-type close to 0.4 µM, which is to the higher end of the range of reported Km values. However, with the proposed range of pCa 882

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dilutions, it is possible to determine any deviations in Km values (i.e., elevations or reductions) relative to a reference value. Problem (Step 30): High variation in activity values. Solution: We propose five possible tactics:

•

•

Check whether the PLB phosphorylation is variable. Strong variation in PLB phosphorylation levels might be due to improper or inconsiderate handling of the mice (Fig. 2A)—stress should be kept to a minimum. We have found that decapitation or other methods (e.g., ether vapor) strongly increase the phosphorylation level of PLB. Pentobarbital injection has no stimulatory effect and is here used to determine the cardiac SERCA2a activity at rest. If PLB phosphorylation is not problematic, check the pH of the reaction as the pH crucially affects the free-Ca2+ concentration. Accurately adjust the pH of the imidazole and EGTA stock solutions. Recheck the pH of the solutions as the pH can vary over time. Prepare stock solutions that can be used for all the experiments, and freeze aliquots of these stock solutions (especially of EGTA and imidazole). Using the same solutions and buffers for all experiments will greatly reduce the variability. Ca2+ dilutions (pCa mix) can be stored in the cold room for 2 mo. Vortex Ca2+ dilutions before use (as precipitates can form). We strongly recommend using a commercially available analytical CaCl2 solution (0.1 M) to prepare the serial dilution as it is extremely difficult to weigh the hygroscopic CaCl2.

•

Ensure that the debris in the homogenate is cleared by spinning for 1 min at 1000g (Step 14) to prevent tissue remains from being transferred into the reaction medium or blocking the pipette tips. Clearance will greatly enhance the reproducibility of the measurements.

• •

Ensure that the filtration device is treated with EGTA before the start. Vortex the reaction mixture after each step.

DISCUSSION

Advanced heart failure is marked by reduced SERCA2a activity (Hasenfuss et al. 1994), which crucially affects the contractility of the heart (Periasamy and Huke 2001). Many mouse models are now available for a detailed study of the SERCA2a regulation in the heart, which is predominantly controlled by PLB and SLN (MacLennan and Kranias 2003). These studies have revealed that, under physiological conditions, the apparent Ca2+ affinity of the pump is meticulously constrained within a narrow range (Vangheluwe et al. 2006a). The most widely used method to measure the rate of ATP-driven Ca2+ uptake into sarcoplasmic reticulum (SR)-derived vesicles involves monitoring the time-dependent increase in net 45Ca2+ uptake in the form of an intravesicular calcium oxalate precipitate (de Meis et al. 1974; Wuytack et al. 1978; Dode et al. 2006). This method has been adapted to measure Ca2+ uptake in crude tissue homogenates of mouse hearts (Ji et al. 1999; Vangheluwe et al. 2006b). Here, we have described this protocol (Ji et al. 1999) in more detail and have shown how the apparent Ca2+ affinity of the Ca2+ pump, which is the most crucial parameter of the SERCA2a activity in vivo (Vangheluwe et al. 2006b; Raeymaekers et al. 2011), can be accurately determined in vitro. The oxalate-dependent Ca2+ uptake provides a rapid and reliable in vitro measurement of SERCA2a-driven Ca2+ transport in individual mouse hearts, providing information on a crucial determinant of cardiac contractility. The method has been used to compare directly wild-type with transgenic or treated hearts in which the activity of SERCA2a can be altered (Ji et al. 1999). However, as the apparent Ca2+ affinity of SERCA2a in vitro depends on the level of phosphorylation of PLB (MacLennan and Kranias 2003), it is important to isolate and treat the hearts carefully Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot076893

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to preserve the endogenous PLB phosphorylation and maintain it in vitro during the course of the experiment. Because of these inherent difficulties, one should not rely solely on the in vitro Ca2+-uptake activities to make firm conclusions on SERCA2a activity in vivo. An altered SERCA2a activity might not necessarily translate into changes in cardiac contractility as compensations might occur at the molecular, cellular or organ level (Vangheluwe et al. 2006b). Thus, only in combination with an analysis of the protein expression (SERCA, PLB, and SLN) and levels of PLB phosphorylation, the cardiomyocyte Ca2+ handling and contractility, together with measurements of in vivo hemodynamics, can one truly evaluate the correlation between SERCA2a activity and in vivo cardiac contractility. In the past, Ca2+ uptake was studied mainly in muscle microsomes, and the amount of murine cardiac tissue could limit preparation of sufficient SR, whereas here Ca2+ uptake is measured in cardiac homogenates (Ji et al. 1999), but note that the protocol can also be used to measure Ca2+ uptake in cardiac or skeletal muscle homogenates or in microsomes of mouse or other species (Ji et al. 1999). As the SERCA2 concentration varies between species, this might affect the results. In general, higher SERCA2a expression is observed in cardiac SR from small in comparison with larger animals (Hove-Madsen and Bers 1993; Bers 2002). Compared with the ATPase method (see High-Throughput Measurement of the Ca 2+Dependent ATPase Activity in COS Microsomes [Vandecaetsbeek et al. 2013]), measuring Ca2+ uptake is a more direct way of assessing SERCA2a activity as, in the ATPase assay, SERCA2a might be (partially) uncoupled. Note that, in our hands, measuring the Ca2+-dependent ATPase activity is possible in cardiac microsomes but not in cardiac homogenates owing to the high ATPase background. SLN is a second SERCA2a inhibitor, homologous to PLB, found in the heart. In the murine heart, SLN is confined to the atria (Minamisawa et al. 2003). Two polyclonal antibodies against SLN have been described that are directed against, respectively, the amino- (Vangheluwe et al. 2005) and carboxyl terminus of SLN (Babu et al. 2007). The carboxy-terminal SLN antibody appears to possess the greater specificity and could be used in this protocol. Finally, with this method, the activity of SERCA2a in hearts of different mouse groups can be compared. It also allows testing various compounds that alter SERCA activity, such as inhibitors or modifiers of the inhibition effected by PLB or SLN (such as protein kinase A, calcium/calmodulin kinase II, phosphatases and antibodies against PLB) (Sham et al. 1991).

RECIPES Homogenization Buffer for Ca2+-Uptake Assay

Reagent K2HPO4 (pH 7.0) NaF Sucrose EDTA (pH 7.0) Dithiothreitol (DTT)

Final concentration 50 mM 25 mM 300 mM 1 mM 0.5 mM

Before use, add per 5 mL homogenization buffer: 5 µL 200 mM PMSF (in ethanol) 50 µL Protease inhibitor cocktail (in DMSO) 50 µL Phosphatase inhibitor cocktail (in DMSO) The protease inhibitor cocktail should be diluted according to the manufacturer’s instructions. Stock solutions can be kept for several months when refrigerated, except DTT, PMSF, protease and phosphatase inhibitor cocktails, which should be kept at −20˚C. 884




Master Mix for Ca2+-Uptake Assay

Reagent

Final concentration

Imidazole–HCl (pH 7.0) KCl NaN3 MgCl2 · 6H2O EGTA (pH 7.0) Potassium oxalate Ruthenium Red [45Ca2+] CaCl2

40 mM 95 mM 5 mM 5 mM 0.5 mM 5 mM 1 µM 1 µCi/mL

Wash Buffer for Ca2+-Uptake Assay

Reagent

Final concentration

EGTA Tris–HCl KCl

1 mM 10 mM 100 mM

The buffer can be pre-prepared and stored at 4˚C for several months.

ACKNOWLEDGMENTS

T.H. is a predoctoral and P.V. a postdoctoral fellow of the Flanders Research Foundation (FWOVlaanderen). This work was financed by the Research Program of the Flanders Research Foundation (FWO-Vlaanderen) G.0646.08 and G.0442.12 and by the Interuniversity Attraction Poles Program P7/13 of the Belgian State, Federal Office for Scientific Technical and Cultural Affairs.

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