SLMAP3 isoform modulates cardiac gene expression and function

Figures

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  Fig 1. Postnatal expression of SLMAP3 in Tg mice. (A) Schematic representation of the SLMAP3 transgene construct comprising the forkhead associated domain (FHA), the leucine zipper coiled-coil region (LZ), and the transmembrane domain 2 (TM2) in frame with 6-myc tag, driven by α-MHC promoter. (B) Number of SLMAP3 transgene copies in hearts. DNA was isolated from hearts of mice and qRT-PCR was performed with appropriate primers to assess copies of Myc-SLMAP3 transgene. (C) SLMAP3 transgene protein expression in Tg mice. Western blot of heart lysate from Wt and Tg mice with anti-myc in high (H), moderate (M), and low (L) SLMAP3-six myc expression, GAPDH was used as loading control. (D) Endogenous SLMAP isoforms in Wt and Tg myocardium. Western blots of SLMAP isoforms in Wt and Tg mice at 5 weeks of age with anti-SLMAP (~35 kDa, ~43 kDa, ~81 kDa, ~91 kDa, and 110 kDa) and anti-myc antibodies (~110 kDa). SLMAP3 image was acquired with a longer exposure of the same membrane. (E) Quantification and fold change of protein expression levels of SLMAP1 (~35 kDa) and SLMAP2 (~43 kDa) isoforms in Tg mice compared to Wt age-matched littermates at 5 weeks of age; Wt corresponds to 1 (100%), n = 3.

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  Fig 2. Histology and cardiac remodeling in SLMAP3 Tg mice. (A) H&E stained paraffin sections of the heart in Wt (a, c) and Tg (b, d) mice at 6 weeks of age. Masson’s Trichrome stained sections of Wt (e) and Tg (f) hearts at 6 weeks of age. Four chamber view sections of the heart were acquired at 12.6x magnification. Pictures at 40x magnification of the four chamber view are representative of random selections throughout the heart. (B) Heart weight (HW) corrected to body weight (BW) at 5 weeks of age acquired during necropsy. Values of mixed groups are represented as fold change of Tg compared to Wt mice. Sex specific groups are represented as fold change compared to Wt females. (C) qRT-PCR of fetal genes: Nppa (ANP), Nppb (BNP), Myh6 (α-MHC), and Myh7 (β-MHC) transcript levels at 5 weeks of age (6 Wt and 8 Tg); *p<0.05.

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  Fig 3. Function of myocardium in SLMAP3 Tg mice. (A) Heart performance and dimensions measured by transthoracic echocardiography in SLMAP3 Tg mice and Wt littermates at 5 weeks of age: ejection fraction (EF), fractional shortening (FS), end-systolic left ventricular volume (LV vol (s)), stroke volume (SV), cardiac output (CO), end-systolic left ventricular internal diameter (LVID (s)). (B) Representative echocardiograms of Wt and Tg mice showing end-diastole and end-systole in B-mode of the short axis with corresponding measurements in M-mode. *p<0.05 Tg compared to age matched Wt littermates.

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  Fig 4. Electrical properties of myocardium in SLMAP3 Tg mice. (A) Representative electrocardiograms of lead II acquired by surface 6-lead ECG and definition of measured intervals in Wt (n = 11) and Tg (n = 8) mice at 5 weeks of age. (B) Quantification of PR interval in Wt and Tg mice measured in lead II. (C) Electrocardiograms acquired in Wt and Tg mice with QRS complexes different from the representative tracings shown in panel A. (D) Quantification of P, R, and S wave amplitudes in Wt and Tg mice measured in lead II. *p<0.05 Tg compared to age matched Wt littermates.

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  Fig 5. Nav1.5 and NCX1 expression in membrane fractions from Tg myocardium. (A) Western blot with anti-Nav1.5 and anti-NCX1 protein expression in the heavy SR fractions from 5 weeks old hearts from Wt and Tg mice (n = 3). (B) Protein quantification of Nav1.5 in heavy SR fraction. (C) Protein quantification of NCX1 in heavy SR fraction; Nav1.5 and NCX1 protein quantifications were normalized to total protein with stain-free and are presented as fold change where Wt = 1 (100%); quantified range of total protein is presented; Tg SLMAP was detected by anti-myc (Myc-SLMAP3).

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  Fig 6. Expression of calcium handling proteins and their phosphorylation in microsomes. (A) Western blot analysis of SERCA2a protein expression in microsomal fractions and total protein acquired by stain-free. (B) Quantification of SERCA2a in microsomal fractions normalized to total protein assessed by stain free technology in Wt (n = 4) and Tg (n = 7) mice. (C) Western blots for total ryanodine receptor 2 (t-RyR2), it’s phosphorylation on serine 2808 (p-RyR2 ser 2808), calsequestrin (CSQ), total phospholamban (t-PLN) and it’s phosphorylation on serine 16 (p-PLN ser16) in microsomal fractions from 5 weeks old Tg and Wt mice hearts. Anti-calreticulin was used as loading control. Tg SLMAP3 was detected by anti-myc (Myc-SLMAP3). (D) Quantification of RyR2, CSQ, and PLN (monomeric) protein expression in Wt and Tg mice. *p<0.05, #p = 0.053, n (Wt) = 5, n (Tg) = 4.

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  Fig 7. mRNA expression of sodium and calcium handling proteins in SLMAP3 and SLMAP1 Tg hearts. (A) mRNA levels of Scn5a (Nav1.5) sodium channel, Slc8a1 (Ncx1) sodium calcium exchanger, Pln, Cacna1c (Cav1.2), and Atp2a2 (SERCA2a) assessed by qRT-PCR in hearts from 5 weeks old Wt (n = 6) and SLMAP3 Tg (n = 8) mouse hearts; (B) mRNA levels of Scn5a in 5 weeks old Wt and SLMAP1-Tg mouse hearts (n = 6). (C) Western blot of Nav1.5 and Tg SLMAP (detected by anti-myc, Myc-SLMAP1) in SLMAP1-Tg and Wt mouse hearts. (D) Quantification of Nav1.5 protein level normalized to α-Tubulin in SLMAP1-Tg and Wt mouse hearts (n = 3). Data are presented as fold change where Wt = 1 (100%), *p<0.05.