Identification of RyR2-PBmice and the effects of transposon insertional mutagenesis of the RyR2 gene on cardiac function in mice

Ethics statement

This study was carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH) (NIH Publication No. 8023, revised 1978). All animal experiments were approved by the Institutional Animal Care and Use Committee of the Navy Medical University (81673348, 1 March 2016). To minimize any suffering of the animals, animals were anesthetized with 1.5% isoflurane before the experiments, and the response of animals during the investigation was monitored to ensure adequate depth of anesthesia. For tissue collection, animals were sacrificed by CO₂ asphyxiation. All animals were housed in a specific animal room on a 12-h light/dark cycle at 22 ± 2 °C and 50–60% humidity, with no more than five per cage.

Animals and reagents

The piggyBac inserted into the RyR2 gene mouse model was provided by the Institute of Developmental Biology, Fudan University (Shanghai, China). The PB transposon was inserted in the first intron of RyR2 on mouse chromosome 13, ENSMUSG00000021313, and the direction of the insertion was opposite to the gene location. All the mice used in this study were in compliance with the Ethics Statement described above.

Collagenase type II was provided by Worthington Biochemical Corporation, USA. Fetal bovine serum was purchased from Gibco, USA. Fluo-4 AM was purchased from Molecular Probes, USA. RIPA lysis buffer was purchased from the Beyotime Institute of Biotechnology, China. Other general agents were obtained from Sangon Biotech, China.

Identification of the RyR2-PBmice

Offspring of the mice with transposons inserted into the RyR2 gene were phenotypically identified by the red fluorescent protein (RFP), a reporter gene carried by RyR2-PBmice. First, we irradiated the mice under an ultraviolet lamp to estimate their genotype by observing the red fluorescence. Then, the RFP images of the isolated cardiomyocytes were obtained by scanning with an Olympus FV1000 confocal microscope at an excitation spectrum of 543 nm.

The RyR2-PBmice strain was further determined by genotyping PCR with the primer pairs GL, GR and PB. β-actin was used as the inner gene when the upstream and downstream primers were 5′-GGCTGTATTCCCCTCCATCG-3′ and 5′-CCAGTTGTACACAATGCCATGT-3′.

Evaluation of cardiac function in mice

RyR2-PBmice and corresponding wild-type mice (WTmice) were assessed for their susceptibility to stimulation-induced ventricular tachyarrhythmia using electrocardiogram (ECG) and heart rate (HR) recordings. Briefly, the mice were lightly anesthetized with 1.5% isoflurane vapor and 95% O₂. The anesthetized mice were placed on a heating pad (27 °C), and needle electrodes were inserted subcutaneously into the right upper limb and left lower abdomen for ECG recording (MPA2000; Alcott Biotech, China). The animal ECG was continuously monitored under anesthesia until the heart rate stabilized. Baseline ECG was recorded for 15 min. For induction of ventricular arrhythmias, the mice were subjected to intraperitoneal injection of a mixture of epinephrine (2 mg/kg) and caffeine (120 mg/kg). ECG and HR were continuously recorded for 30 min after the injection of epinephrine and caffeine.

After the completion of monitoring, the mouse blood sample was collected at the tip of the heart. After resting for half an hour in a 1.5 ml EP tube, the sample was centrifuged and the heart injury-related enzymes were measured with an automatic biochemical analyzer (AIRONE-200puls, Crony Company, Italy).

Myocardial cell calcium capacity test by the confocal microscopy

Single cardiomyocytes were isolated from 3-month-old RyR2-PBmice and WTmice by enzymatic digestion (Fares et al., 1998). Briefly, the hearts were excised from heparinized and deeply anaesthetized mice, cannulated and mounted on a Langendorff apparatus. After a digest perfusion for 8–10 min with perfusion buffer (mM: 10 HEPES, 0.6 Na₂HPO₄, 113 NaCl, 4.7 KCl, 12 NaHCO₃, 0.6 KH₂PO₄, 1.2 MgSO₄ ⋅7H₂O, 10 KHCO₃, 30 taurine, 10 2,3-butanedine monoxime, 5.5 glucose, pH 7.46) containing 773 U/ml collagenase type II (Worthington, USA), the ventricular tissue was cut into small pieces and gently stirred in stopping buffer containing perfusion buffer, 10% fetal bovine serum and 12.5 µM CaCl₂ for 10–15 min. Then, the upper cell suspension was transferred to a 25 ml beaker, and Ca²⁺ was reintroduced to a final concentration of 1 mM. The isolated cardiomyocytes at the bottom were added to glass coverslips precoated with 50 mg/ml laminin and loaded with 5 mM Fluo-4 AM (Molecular Probes, Eugene, OR, USA) in bath solution containing (in mM) 135 NaCl, 4 KCl, 2 CaCl₂, 1 MgCl₂ ⋅H₂O, 10 HEPES, 1.2 NaH₂PO₄ ⋅H₂O and 10 glucose for 20 min at room temperature. Following the monitoring of Ca²⁺ at rest, cells were excited by caffeine stimulation. Confocal line scanning (512 pixels and 1 ms per line) was performed along the longitudinal axis of cells for 10 s using an Olympus FV1000 confocal system (Olympus Corporation, Tokyo, Japan). The fluorescence values (F) were normalized by the basal fluorescence (F₀) in order to obtain the fluorescence ratio (F/F₀).

Cardiac pathological examination

The mouse heart was excised and washed in ice-cold PBS (0.1 M phosphate) after evaluation of cardiac function. For histological analysis, the hearts were fixed with a 10% formalin solution and stained with hematoxylin and eosin for microscopy examination (ECLIPSE 55i Nikon, Tokyo, Japan). The heart samples for transmission electron microscopy observation were fixed in 1% potassium ferrocyanide reduced O_SO₄, dehydrated through graded ethanol, and coated with epoxy resin. Ultrathin slices were stained with saturated uranium acetate and silver citrate and viewed using a transmission electron microscope (H-600, Hitachi, Japan).

Measurement of ATP in the heart tissue

We used a commercial kit (Beyotime Institute of Biotechnology, Shanghai, China) to quantify the content of adenosine triphosphate (ATP) according to the manufacturer’s protocol. In brief, the samples of heart tissues (each approximately 10 g) were homogenized and centrifuged at 1, 200 × g for 10 min at 4 °C. The supernatants were subjected to immediate measurement of ATP levels.

RT-PCR and Western blotting

For the quantitative RT-PCR assay, the total RNA was isolated from the heart tissues of RyR2-PBmice and WTmice by using TRIzol reagent (Invitrogen, USA), and the iScript reaction (Bio-Rad, USA) was used to synthesize the cDNA. Quantitative RT-PCR with iTaq Universal SYBR Green Supermix (Bio-Rad, USA) was performed on a StepOne Real-Time PCR System (Applied Biosystems, USA) to determine gene expression using the relative standard-curve method. β-actin was used as an internal control. The primer sequence information is shown in Table 1 below.

For Western blotting analysis, the total proteins were extracted from the frozen hearts with RIPA lysis buffer according to the manufacturer’s instructions and quantified with the BCA™ Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Antibodies to RyR2 (1:1000, ab196335; Abcam, Cambridge, UK), RFP (1:1,000, ab62341; Abcam), GNB3 (1:2,000, ab154866; Abcam), COX (1:1,000, ab33985; Abcam), LGR5 (1:2,000, ab75732; Abcam), PMCA (1:1,000, ab2825; Abcam), Natriuretic Peptide Receptor B (NPPB, 1:2,000, ab139188; Abcam) and UCP (1:1,000, ab10985; Abcam) were used. Briefly, equal amounts of samples were subjected to SDS-PAGE and then transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with Tris-buffered saline (TBS-T, 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.02% Tween 20) and then blocked with 5% nonfat milk for 1 h at room temperature. After this, the membranes were incubated in the appropriate primary antibodies overnight at 4 °C, followed by HRP-conjugated secondary antibodies (1:1,000, #5127; Cell Signaling Technology, Danvers, MA, USA) at room temperature for 1 h. To control equal loading of total protein in all lanes, blots were stained with rabbit anti-β-actin antibody (1:1,000, #4970; Cell Signaling Technology, Danvers, MA, USA) for equal loading of proteins. After repeated washing, membranes were incubated with an enhanced chemiluminescence system (ECL, Amersham) according to the manufacturer’s instructions followed by visualization with X-ray film. Quantitative analysis was carried out with NIH ImageJ software.

Statistics

All the quantitative data are expressed as the mean ± SD. The graphs were built using GraphPad Prism 5 for each experiment. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used, and a statistical significance was indicated by P < 0.05.

Identification of the RyR2-PBmice

Based on the information we obtained from the PBmice mutation database, the RyR2-PBmice were a strain of heterozygous mouse transposed by a PB transposon element with an RFP gene, so when illuminated by ultraviolet (UV) light, the red fluorescence could be observed in the heterozygous RyR2-PBmice. We found that all surviving individuals in the third generation of breeding showed the same appearance under a fluorescent lamp (Fig. 1A). However, the heterozygous RyR2-PBmice, but not homozygous WTmice, showed visible red fluorescence under UV illumination (Fig. 1B). The results observed in the isolated ventricular myocytes were consistent with those in vivo: the ventricular myocytes from RyR2-PBmice but not from WTmice showed red under RFP excitation.

The genotypes of WTmice and RyR2-PBmice were further identified with the PCR assay. In this procedure, an insertion line carrying the PB transposon was mapped in the RyR2 transposon allele (Fig. 2A). GL, GR and LB primers were used to explore the transposition behavior of PB in the mice. Due to the transposon insertion, the 655 bp and 823 bp PCR products were detected in the RyR2-PBmice, while only an 823 bp product was detected in the WTmice (Fig. 2B).

Effects of transposon insertional mutagenesis in the RyR2 gene on cardiac function

We monitored the ECG and HR of the RyR2-PBmice before and after the injection of pharmacological triggers (caffeine and epinephrine) to assess the effects of insertional mutagenesis in the RyR2 gene by PB transposon on cardiac function. Unexpectedly, we found that caffeine and epinephrine did not induce abnormal cardiac function in either RyR2 mutant mice or their WT littermates at 2–3 months of age (Fig. 3). We further detected cardiac function-related enzymes to evaluate heart injury in RyR2-PBmice. Similarly, we did not find any significant difference in phosphocreatine kinase (CK), lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) indexes between the RyR2-PBmice and WTmice (Fig. 4).

Effects of PB insertion on intracellular Ca²⁺ homeostasis in cardiomyocytes

To evaluate the influence of PB insertion on intracellular Ca²⁺ homeostasis in RyR2-PBmice, we measured caffeine-evoked [Ca²⁺]_i transients using the Ca²⁺ indicator Fluo-4 AM. Figures 5A and 5B show the corresponding [Ca²⁺]_i transients by caffeine stimulation in cardiomyocytes obtained from WTmice and RyR2-PBmice, respectively. The amplitude of [Ca²⁺]_i transients (F/F0) was lower in RyR2-PBmice cardiomyocytes than in WTmice. We further measured the isoproterenol (ISO)-evoked [Ca²⁺]_i transients and found that the [Ca²⁺]_i capacity was also significantly reduced in the RyR2-PBmouse cardiomyocytes.

Mitochondrial structure disorder in cardiomyocytes

Histological analysis of heart sections stained with hematoxylin and eosin revealed that the cardiac sarcomere was well-arranged and no obvious lesions were observed in the myocardium from both WTmice and RyR2-PBmice (Fig. 6). To investigate the ultrastructure of the mitochondria, transmission electron microscopy was performed on cardiac muscle tissue (Fig. 7). Compared with the WTmice, significant mitochondrial swelling and focal dissolution of mitochondrial cristae occurred in RyR2-PBmice. These results suggested that while no obvious histological changes occurred in the myocardial structure of RyR2-PBmice, PB transposons inserted into the RyR2 gene disrupted the ultrastructure of myocardial mitochondria.

ATP concentration in heart tissues

To investigate whether the myocardial mitochondrial structure affected cardiac function, we examined the concentration of ATP in myocardial tissue to evaluate its energy supply. The results showed that the ATP concentration in RyR2-PBmouse heart tissue was significantly reduced compared with WTmice (Fig. 8), indicating that the piggyBac inserted into the RyR2 gene could interfere with the energy metabolism level of the mitochondria.

RT-PCR and Western blotting

To examine the expression level of the RyR2 and genes from its related signaling pathway, real-time quantitative PCR (qPCR) was applied to quantify the 3′-end transcripts with primer pairs located on these genes. The mRNA levels of a variety of genes were decreased, including RyR2 (Fig. 9A), Gnb3 (Fig. 9B), Cox8b (Fig. 9C), Cdh11 (Fig. 9E), Micu3 (Fig. 9F), mt-Co2 (Fig. 9G), mt-Atp6 (Fig. 9H), mt-Co3 (Fig. 9I), Nppb (Fig. 9J) and Ucp3 (Fig. 9K), whereas the Gpr37l level was increased (Fig. 9D). Gnb3 and Gpr37 l1 are related to guanine nucleotide binding proteins, which are involved as modulators or transducers in various transmembrane signaling systems and are important in regulating calcium channels. Cox8b, Micu3, mt-Co2, mt-Atp6, mt-Co3 and Ucp3 are responsible for the oxidative phosphorylation and ATP synthesis in the mitochondrial. Nppb, the B type natriuretic peptide, is a hormone secreted by cardiomyocytes in the heart ventricles in response to stretching caused by increased ventricular blood volume. These genes are essential for normal cardiac and mitochondrial function, and the reduction of these genes at the transcriptional level also suggested that the insertion mutation of RyR2 could result in mitochondrial energy metabolic disorders.

Western blot analysis (Fig. 10) showed that there was no significant difference in the protein levels of RyR2, PMA and G protein-coupled receptors between WTmice and RyR2-PBmice, while the expression levels of small G protein and cytochrome C oxidase associated with the RyR2 signal pathway significantly decreased.