Generation of Knock-In Mice with the Ryr2 N4103K Mutation

Supplemental materials

Detailed Methods

Generation of knock-in mice with the RyR2 N4103K mutation.

We isolated the template clone containing exon 87-93 from C57BL/6J mouse genomiclibrary. The 3.7kbp SacII-10200-13913 XmaI fragment (Short fragment) from exon87 to 90 was amplified by PCR using the template. To introduce a point mutation N4103K, the 900bp 3’-phosphorylated ClaI-13914-14816 fragment of the exon 90 (Long 1 fragment) and the 5.7Kb 5’-phosphorylaed 14817-N4103K-20497-SalI fragment (Long 2 fragment) isolated from exon 89 to 93 were connected at their blunt ends (Long fragment). The Short fragment inserted into the pBS-LNL(+) vector at SacII-XmaI sites to introduce LoxP-Neo at the 3’-end, and the Long fragment inserted into the pBS-DTA vector at ClaI-SalI sites to introduce the diphtheria toxin A(DTA) fragment for negative selection at the 3’-end. Afterinsertion of the Short- LoxP-Neo fragment into the Long-DTA vector at SacII-ClaI sites, the constructed targeting vector was linearized with SacII andtransfected into the embryonic stem (ES) cells byelectroporation. The targeting vector, which was constructed as above, was linearized with SacII andtransfected into the embryonic stem (ES) cells byelectroporation. Positive andnegative selections were performed using Geneticin and diphtheria toxin A (DT-A) fragment,respectively. About 100 clones were selected and analyzed by PCR using the neo P1 (5’-CAGTGAAACTGTGGTCATTAGAGAG-3’) and the exon primer (5’-CATATTTTTGTAGCTTAGGCATTGG-3’) and then confirmed by sequencing and Southernblot analysis. Theselected ES cells were injected into the blastocysts of BALB/c mice and chimeric animals wereobtained. The chimeric mice gave offspring with germ-line transmission, and miceheterozygous for the targeted RyR2+/RyR2N4103K-neo were established (F1 mice). The hybrid line of knock-in mice was established by mating with CAG-Cre mice to remove the neo cassette. The genotypes of the F2 (RyR2+/RyR2N4103K) generations were determined by PCR analysis of genomic DNA using the forward primer (5’ -ATCTTGCCAATTATCTACCTGTTTG-3’) and the reverse primer (5’ -AGAAATGAAGAGATTACCCATAAGC- 3’).

Histology.

Hearts from N4103K/+ KI and WT mice, aged 16-22 weeks, were fixed with 10%formalin. A complete, full-circumferential section, at the level of the two left ventricularpapillary muscles, was selected for morphometric analysis. Hematoxylin-Eosin stains was performed for each section of the ventricle.

Echocardiography.

Cardiac function was analyzed by a HDI-5000 ultrasound machine (Philips, Netherlands)equipped with a 15-MHz probe. KI and WT mice were initially anesthetized with4-5% isoflurane (mixed with oxygen) and maintained with 1-2% isoflurane duringechocardiography.Heartrate (HR), left ventricular end-diastolicdiameter (LVEDD), left ventricular endsystolicdiameter (LVESD), intraventricularseptum diastolic thickness (IVSD), leftventricular posterior wall diastolic thickness(LVPWD), left ventricular fractionalshortening [LVFS: (LVEDD/LVESD)/LVEDDx100)] were measured

Surface electrocardiogram

ECG was monitored in a conscious state for N4103K/+ KI and WT mice by using ECGtelemetry. Briefly, transmitters (Data Sciences International, St. Paul, MN) were implanted in theback space with subcutaneous electrodes in a lead II configuration. Telemetry was recorded in a anesthetic state at baseline and after the injection of epinephrine (1 mg/kg of bodyweight i.p.) and caffeine (120 mg/kg of body weight i.p.) and monitored for 30 minutes in a subset ofKI (n=7) and WT mice (n=5).

Expression and purification of CaM and Gly-Ser-His (GSH)-CaM

The mammalian CaM (mCaM) cDNA was kindly provided by Dr Zenon Grabarek (Boston Biomedical Institute, Boston, MA). Human CaM cDNA was polymerase chain reaction-amplified with oligonucleotide primers designed to include two restriction enzyme sites (the forward primer 5′-ACACAGGGGATCCCATATGGCTGAC-3′and the reverse primer 5′-CAAGCTTGGCTCGAGTCACTTTGC-3′). The cDNA was inserted into a pGEX4T-1 vector. The expression vector was transformed into DH5 Escherichia coli (Nippongene). The strain was pre-incubated with lysogeny broth (LB) containing ampicillin for 16 h at 30°C followed by 2 h of incubation with 10-times the volume of LB with ampicillin at 37°C.

Isolation of cardiac cardiomyocytes

Cardiomyocytes were isolated from the mouse hearts as described previously [1-4]. In brief, mice were anaesthetized with pentobarbital sodium (70 mg/kg of body weight, i.p.), intubated, and ventilated with ambient air. An incision in the chest was made, and the heart was quickly removed and retrogradely perfused with a collagenase-free buffer via the aorta under constant flow. The left ventricular myocardium was minced with scissors in a fresh collagenase-containing buffer and the rod-shaped adult mouse cardiomyocytes were prepared by retrograde perfusion of the hearts with 95% O2/5% CO2-bubbled minimal essential medium (Sigma, St Louis, MO, USA) supplemented with 50 μmol/L [Ca2+], 0.5 mg/mL collagenase B, 0.5 mg/mL collagenase D, and 0.02 mg/mL protease type XIV. The Ca2+ concentration was then gradually increased to a final concentration of 1 mM by changing the incubation medium (50 μM, 100 μM, 300 μM, 600 μM, and then 1 mM). The isolated mouse cardiomyocytes were transferred to laminin-coated glass culture dishes, and incubated for a few hours at 37°C in a 5% CO2/95% O2 atmosphere.

Immunocytochemistry analysis of endogenous RyR2-bound CaM

Isolated cardiomyocytes were fixed with 2% paraformaldehyde for 2 min and permeabilized with 20C methanol. Then, the cardiomyocytes were incubated overnight at 4°C with the anti-CaM antibody (EP799Y, abCaM) and anti-RyR antibody (Sigma-Aldrich, C3-33) in 1% bovine serum albumin and 0.5 % Triton X-100, followed by labelling with an Alexa488-conjugated goat anti-rabbit and an Alexa633-conjugated goat anti-mouse secondary antibody. The sarcomere-related periodical increase in the Alexa633 and Alexa488 fluorescence intensity from baseline was integrated with respect to the longitudinally selected distance (~25 μm) and then the value was divided by the distance. Then mean value of one sarcomere-related increased fluorescence intensity was calculated as the arbitrary amount of RyR and RyR-bound CaM.

Measurement of the binding affinity of fluorescently labelled CaM to RyR2 in saponin-permeabilized cardiomyocytes

The fluorescently labelled CaM with HiLyte Fluor 647 (AnaSpec Inc., FremontCA, USA) (HiLyte -CaM), was introduced into the saponin-permeabilizedcardiomyocytes under the same conditions as described below for the Ca2+ spark measurements. Then, the distribution of localized CaM was determined by densitometric measurement of HiLyte-CaM fluorescence. Briefly, the fluorescently labelled cardiomyocytes were laser-scanned with the confocal microscope (LSM-510, Carl Zeiss, numerical aperture, 1.3; excitation at 633 nm; emission at 640 nm). The sarcomere-related periodical increase in the HiLyte Fluor 647 fluorescence intensity from baseline was integrated with respect to the longitudinally selected distance (~25 µm) and then the value was divided by the distance. The mean value of one sarcomere-related increased fluorescence intensity was calculated as the arbitrary amount of RyR-bound CaM.

Analysis of Ca2+ sparks and sarcoplasmic reticulum (SR) Ca2+ content

Ca2+ sparks were measured in saponin-permeabilized cardiomyocytes as previously described using a laser-scanning confocal microscope (LSM-510, Carl Zeiss) [1, 4, 5]. In brief, ventricular myocytes were superfused with a relaxing solution containing 0.1 mM EGTA, 5 mM ATP, 10 mM HEPES, 150 mM K-aspartate, 0.25 mM MgCl2, and 10 mM reduced glutathione at 23°C. The sarcolemma was permeabilized by treatment with saponin (50 μg/mL) for 30 s. After permeabilization, cardiomyocytes were placed in an internal solution composed of 0.5 mM EGTA, 10 mM HEPES, 120 mM K-aspartate, 5 mM ATP, 1 mM free [Mg2+], 10 mM reduced glutathione, 40 nM free [Ca2+] (calculated using MaxChelator, http://www.stanford.edu/~cpatton/webmaxcS.htm), 5 U/ml creatine phosphokinase, 10 mMphosphocreatine, 4% dextran (Mr: 40,000), and 0.02 mM Fluo-4; pH 7.2. Fluo-4was excited by 488 nm laser lines, and the fluorescence intensity was acquired at excitation wavelengths of 505-530 nm. Ca2+ spark images were obtained from permeabilized ventricular myocytes in the presence of the Ca2+/CaM-dependent protein kinase II (CaMKII) inhibitor KN-93 (1 µM) and okadaic acid (1 µM).

Data were analyzed with SparkMaster, an automated analysis program that allows for rapid and reliable spark analysis [6]. To assess the SR Ca2+ content, caffeine (10 mM) was rapidly perfused to discharge the SR-loaded Ca2+.

Monitoring of Ca2+ transients incardiomyocytes.

Isolated ventricular myocytes were incubated with 20 μM Fluo-4 acetoxymethyl ester for 30 min at room temperature and washed twice with Tyrode's solution. All experiments were conducted at 35°C. Intracellular Ca2+ measurements with cells stimulated by a field electric stimulator (IonOptix, MA, USA) were performed with a laser-scanning confocal microscope (LSM-510, CarlZeiss) and fluorescent digital microscopy (BZ9000, Keyence, Japan). The relative occurrence of spontaneous Ca2+ release upon cessation of stimulation at 1, 2, 3, 4, and 5 Hz was measured in WT and KI cardiomyocytes [4].

Peptide-peptide interaction measurement

Binding of DP4089 or DP4089mut to CaMBP in the presence or absence of CaM was detected by using a27-MHz Quartz Crystal Microbalance (QCM: Initium Inc, Japan), which is a highly sensitivemass-measuring apparatus. The QCM Au electrode was coatedwith CaMBP and immersed in a solution (500 μL) containing(in mM 150NaCl, 20MOPS, 0.01 Ca2+ (pH 7.2). The amount of peptidebinding was determined from the frequency changes due to changesin mass on the electrode (with sensitivity on the order of subnanogram)on injection of a small volume (2 to 5 μL) of solutioncontaining DP4089, DP4089mut or CaM. No significant nonspecificbinding of DP4089, DP4089mut or CaM to the Au electrode was detected.

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2. Yamamoto T, Yano M, Xu X, Uchinoumi H, Tateishi H, Mochizuki M, Oda T, Kobayashi S, Ikemoto N, Matsuzaki M. Identification of target domains of the cardiac ryanodine receptor to correct channel disorder in failing hearts. Circulation. 2008;117:762-772.

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Legends to supplementalfiguresand table

Supplemental Figure 1.

Structural characterization of hearts from WTand N4103K/+ KI mice.

Representativeimages of hematoxylin/eosin-stainedhearts from WT and KI mice.

Supplemental Figure 2.

Functional characterization of hearts from WTand N4103K/+ KI mice.

Summarizeddata of heartrate (HR), left ventricular end-diastolicdiameter (LVEDD), left ventricular endsystolicdiameter (LVESD), intraventricularseptum diastolic thickness (IVSD), leftventricular posterior wall diastolic thickness(LVPWD), left ventricular fractionalshortening [LVFS: (LVEDD/LVESD)/LVEDDx100)]. N indicates the number ofmice examined.

Supplemental Figure 3.

ECG characteristics of WTand N4103K/+ KI mice.

There was no significant difference in resting ECG characteristics between WT (n=5) and KI (n=5) mice.