Enhancing atrial‐specific gene expression using a calsequestrin cis‐regulatory module 4 with a sarcolipin promoter

Figures

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  Figure 1. Sarcolipin expression increases in heart failure mouse samples. Sarcolipin expression was compared in heart failure mouse samples from 4, 8 and 16 weeks post TAC surgery

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  Figure 2. Generation of AAV constructs with various promoters. (A) Schematic depiction of CMV, SLN and CRM4.SLN promoters with luciferase or EGFP as a reporter gene. (B) The full 1289‐bp sarcolipin promoter sequence. (C) The full 207‐bp CRM4 sequence

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  Figure 3. A luciferase assay confirmed CRM4 enhanced‐atrial specificity and minimized off‐target expression. (A) A luciferase assay was conducted 24 hours post incubation in HL1 cells. Gene expression of control and luciferase containing CMV, SLN and CRM4.SLN vectors was measured. (B) In vivo gene expression was observed 3 weeks post tail vein delivery of control and luciferase containing CMV, SLN and CRM4.SLN vectors. A luciferase assay was conducted to determine bio‐distribution. Luciferase activity was normalized to background values. Statistical significance was measured with a two‐tailed Student's t‐test and significant differences are indicated: *p < 0.05, **p < 0.01, ***p < 0.005. Bar graphs represent the mean ± SD (n = 4)

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  Figure 4. Atrial specificity and expression by CRM4 were validated by an alternative reporter, EGFP. In vivo gene expression was observed 3 weeks post tail vein delivery of control and EGFP containing CMV, SLN and CRM4.SLN vectors. Western blot analysis was conducted to determine bio‐distribution with antibody EGFP and normalized with tubulin

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  Figure 5. mRNA analysis validated EGFP gene expression profiles. In vivo gene expression was observed 3 weeks post tail vein delivery of control and EGFP containing CMV, SLN and CRM4.SLN vectors. qRT‐PCR analysis was conducted to determine bio‐distribution with EGFP and normalized with 18S. Statistical significance was measured with a two‐tailed Student's t‐test and significant differences are indicated: *p < 0.05, **p < 0.01, ***p < 0.005. Bar graphs represent the mean ± SD (n = 4)

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  Figure 6. Visualization of biodistribution. In vivo gene expression in the atrium and ventricle of control and EGFP containing CMV, SLN and CRM4.SLN vectors was observed by fluorescence imaging using a confocal microscope at 200× magnification. Bright field, DAPI, EGFP and merged images were taken under same exposure and normalization settings