Generation of transgenic mice
The generation and phenotypic characterization of the SMN-deficient transgenic SMNΔ7 mouse line (SMNΔ7;SMN2;Smn−/−) has been described previously [22]. Briefly, the SMNΔ7 mouse line is a triple mutant model with a double copy of SMA cDNA lacking exon 7 and two copies of the human SMN2 on an Smn−/− background. SMNΔ7 mouse Transgenic and unaffected littermate mice (postnatal day 5 (P5) and 10 (P10), both male and female) were used in the present study. All procedures complied with the standards for the care and use of animal subjects as stated in the Guide or the Care and Use of Laboratory Animals (NIH publication No. 85 − 23, revised 1996) and all protocols were approved by the IACUC at the Uniformed Services University of the Health Sciences.
RNA extraction and quantification: Tissue (50–100 mg) was homogenized in 1 ml TRIzol reagent (Thermo Fisher, cat 15596018), and total RNA was isolated and converted to cDNA as previously described [23]. Gene specific Taqman primer and probe sets were purchased from Applied Biosystems: atrial natriuretic peptide (ANP; Nppa, Mm01255748_g1), brain natriuretic peptide (BNP, Nppb, Mm01255770_g1), skeletal muscle actin (Acta1, Mm00808218_g1), SERCA2 (Atp2a2, Mm01201431_m1), hypoxanthine guanine phosphoribosyl transferase (Hprt, Mm01318743_m1) and glyceraldehyde 3-phosphate dehydrogenase (Gapdh, Mm01180221_g1). qRT-PCR reactions were performed in triplicate using the StepOnePlus™ Real-Time PCR System (Applied Biosystems). The following primers were purchased from Integrated DNA Technologies: SERCA2, 5′-TGAGACGCTCAAGTTTGTGG-3′; SERCA2a, 5′-ATGCAGAGGGCTGGTAGATG-3′; SERCA2b, 5′-ACAAACGGCCAGGAAATG-3′; and GAPDH, 5′-GCATGGCCTTCCGTGTTC-3′, 5′-ATGTCATCATACTTGGCAGGTTTC-3′. The level of each transcript was quantified by the threshold cycle (Ct) method using Gapdh and Hprt as endogenous controls.
Values were normalized to the mean of the unaffected group for each gene, which was assigned as 1.
Transcriptome profiling by RNA sequencing
Total RNA was quantified via a fluorescence dye-based methodology (RiboGreen, Thermo Fisher, cat R11490) on a Spectramax Gemini XPS plate reader (Molecular Devices, Mountain View, CA). RNA integrity was assessed using automated capillary electrophoresis on a Fragment Analyser (Advanced Analytical Technologies, Inc, Santa Clara, CA). Total RNA input of 200 ng was used for library preparation using the TruSeq Stranded mRNA Library Preparation Kit (Illumina, San Diego, CA, cat 20020594). Sequencing libraries were quantified by PCR using KAPA Library Quantification Kit for NGS (Roche, Wilmington, MA, cat KK4854) and assessed for size distribution on a Fragment Analyser. Sequencing libraries were pooled and sequenced on a NextSEq. 500 Desktop Sequencer (Illumina) using a NextSEq. 500 High Output Kit v2 with paired-end reads at 75 bp length. Raw sequencing data was demuxed using bcl2fastq2 Conversion Software 2.17 before alignment using TopHat Alignment v1.0 and differential expression analysis using Cufflinks Assembly & DE v1.1.0 on BaseSpace Onsite (Illumina). Functional enrichment analysis was performed using the PANTHER Classification System. Significantly overrepresented gene ontology biological processes at p < 0.01 were adjusted using a Bonferroni correction for multiple testing.
Western blots: Fresh ventricular tissue was homogenized (Polytron) in RIPA lysis buffer (Sigma, cat R0278). Isolated proteins were separated by SDS-PAGE (4–15%, BioRad, cat 456–1084) and transferred onto PVDF membranes (BioRad, BR20191004). Blots were probed with the following specific antibodies: anti-SERCA2a antibody (1:1000, Cell Signaling Technology, cat 9580), anti-SMN (1:1000, BD Transduction Laboratories, cat 610647), anti-α-actin (1:5000 mybiosource.com, MBS477269) anti-BNP (1:1000, Abcam, cat ab236101) and GADPH (1:5000, Abcam, cat ab8245). Bound antibodies were detected with SuperSignal West Dura ECL substrate (Pierce, cat 34075).
Cell contractility and calcium transient measurements in mouse cardiomyocytes:
Unloaded cell shortening and intracellular calcium (Ca2+) transients were measured in freshly isolated ventricular myocytes, prepared as described previously [24, 25]. Isolated myocytes were transferred into a recording chamber mounted on an Olympus X51 inverted microscope and superfused with normal Tyrode solution (composition in mM: NaCl, 137; KCl, 5.4; NaH2PO4, 0.16; glucose, 10; CaCl2, 1.8; MgCl2, 0.5; HEPES, 5.0; NaHCO3, 3.0; pH 7.3–7.4.). All experiments were performed at room temperature. Video images were acquired using a Myocam camera (IonOptix). Cells were field stimulated at 1 Hz in all experiments.
In experiments aimed at measuring intracellular Ca2+, isolated cells were incubated in Wittenberg Isolation Medium (WIM, composition in mM: NaCl, 116; KCl, 5.3; NaH2PO4, 1.2; glucose, 11.6; MgCl2, 3.7; HEPES, 20; L-glutamine, 2.0; NaHCO3, 4.4; KH2PO4, 1.5; 1X essential vitamins (GIBCO cat 12473-013);1X amino acids (GIBCO cat 11120-052); pH 7.3–7.4.) solution containing fluo-3-AM (1 µM) for 30 minutes at room temperature. Fluo-loaded cells were transferred into the recording chamber and perfused with normal Tyrode solution supplemented with 500 µM probenecid to inhibit dye export. Cells were stimulated to contract at 1 Hz in all cases and fluorescence was captured with a photomultiplier tube (IonOptix). Intracellular Ca2+ concentration ([Ca2+]i) was estimated using the Maravall equation [26]:
Where Kd was assumed to be 600 nM [27] and Rf = Fmax/Fmin. Fmin and Fmax were measured in each cell by incubating the cell in modified Tyrode solutions supplemented with 20 mM 2,3 butadione monoxime, 10 mM A23187 and either 10 mM EGTA, or 100 mM CaCl2, respectively.
Cell Lines: To study the effects of SMN-deficiency on human cardiomyocytes, we utilized iPSCs from a spinal muscular atrophy patient (GM23240; Coriell). These iPSCs were reprogrammed from fibroblasts using lentiviral constructs encoding OCT4, SOX2, NANOG and LIN28. NCRM-1 iPSCs were used as a control line and obtained from XCell Sciences (Novato CA). NCRM-1 iPSCs were obtained from XCell Sciences and reprogrammed by episomal plasmid from CD 34 + human cord blood cells. To grow and maintain iPSC lines, standard 6-well tissue culture plates were coated with growth factor reduced Matrigel (Corning; cat 354277 ) diluted 1:100 in DMEM/F12 (Gibco, 11330-032) same day as iPSC plating. Frozen stocks of iPSCs were thawed and plated on Matrigel coated plates in TeSR E8 (StemCell, cat 05990) supplemented with ROCK inhibitor (Y-27632, 10uM, Tocris, cat 1254). After 24 hours, media was replaced with TeSR E8 (without ROCK inhibitor) and culture media was replaced daily until fully confluent. iPSC lines were passaged using 0.5 mM EDTA (Life Technologies, cat 15575-038) in PBS without CaCl2 and MgCl2 (Hyclone, Slt30256.01). Cells were maintained at 37 °C, 5% CO2.
Generation and Maintenance of Cardiomyocytes
We began the cardiomyocyte differentiation protocol when cells were 80% confluent using the protocol developed by Feaster et al [28]. Briefly, differentiation was initiated (day 0–2) by replacing TeSR E8 medium with RPMI 1640 medium (Lonza, cat 12-702F) supplemented with B27 (minus insulin, Gibco, cat A1895601) and CHIR99021 (6 µM, LC Laboratories, cat C-6556), a GSK3 inhibitor. On days 3–4, CHIR99021 was removed and replaced with RPMI 1640 medium supplemented with B27 (minus insulin) and IWR-1 (500 µM, Sigma, cat I0761), a Wnt signaling inhibitor. On days 5–9, cells were maintained in RPMI 1640 medium supplemented only with B27 (minus insulin). From days 10–15, a metabolic selection protocol was employed using RPMI 1640 without glucose (Life Technologies, cat 11879) plus B27 without insulin. Following metabolic selection, cells were maintained in RPMI 1640 supplemented with B27 (Gibco, cat 17504-044) and 1% pen strep (Gibco, cat 10378-016). Beating cardiomyocytes were fed daily until day 20 when functional assays were carried out as described below.
Transfection
To increase SMN expression in patient iPSC-derived cardiomyocytes, we transfected cells with SMN-GFP cDNA plasmid (1 µg) using Lipofectamine 3000 (Invitrogen, cat 100022052) and P3000 (Invitrogen, cat 100022058) for 48 hours according to the manufacturer protocols. Transfection was performed at day 18 of differentiation. Genartion of SMN-GFP plasmid was previously described [29] and a gift from Greg Matera (University of Carolina at Chapel Hill). To reduce SMN expression in control iPSC-derived cardiomyocytes, cells were transfected with 40 pmol of pre-validated siRNA (IDT, cat 37206943) and RNAi Max (Life Technologies, cat 56532) per manufacturer protocol at the 18 day time point. Opti-MEM (Gibco, cat 11058-021) diluent was used in both protocols. Successful transfection was confirmed in Western Blot analysis.
Flow cytometry: SMA and Control patient iPSC-derived cardiomyocytes were dissociated using TrypLE (Gibco, cat 12605-010). Disaggregated cells were centrifuged and resuspended in fixation buffer (Biolegend, cat 554655) for 15 minutes at room temp, washed 3 times in 1X permeabilization wash buffer (Biolegend, cat 421002, and resuspended in permeabilization wash buffer containing FITC conjugated anti- Cardiac Troponin (1:20, Miltenyi Biotec, cat 130-106-689) for 1 hour at room temp protected from light. Cells were read in cell staining buffer (Biolegend, cat 420201) on BD Accuri C6 Flow Cytometer CFlow Plus software. Unstained cells were used as a negative control.
Calcium Imaging: SMA and Control patient iPSC-derived cardiomyocytes wells were washed with PBS and incubated in normal Tyrode’s solution (Composition in mM: NaCl, 137 mM; KCl, 5.4 mM; NaH2PO4, 0.16 mM; glucose, 10 mM; CaCl2, 1.8 mM; MgCl2, 0.5 mM; HEPES, 10.0 mM; NaHCO3, 3.0 mM; pH 7.3–7.4.) supplemented with cell permeant Fluo-4 AM, fluorescent Calcium indicator (1 µM, Invitrogen, cat F14201) and probenecid (500 µM, Sigma, cat P8761). Cells were incubated for 15 min at 37 °C, 5% CO2. Calcium transients were recorded on a Nikon eclipse Ti2 inverted microscope equipped with a large view CMOS camera on a 20x objective with NIS Elements AR software (Nikon). Decay at T10, T50, T75, T90 and T100 were analyzed using Clampfit software (Axon).
Reagents
A complete list of reagents, primer sequences with source information can be found in Supplemental Table 1.
Data analysis
All data were analyzed using IonWizard, Clampfit, and Microsoft Excel software and, except where noted, results are presented as means ± SEM (standard error of the mean). In all cases P < 0.05 was considered significant. Statistical tests used and resultant p-values are given in the Figure legends. Statistical analysis was performed in GraphPad Prism 7.