Fig 1.
Striated muscle specific deletion of LPL leads to hypertriglyceridemia.
A. Quantitative PCR for mouse LPL (mLPL) mRNA expression in skeletal muscle (SKM) or heart (HRT) from wild-type (WT) and mck-Cre;lpl -/- (cKO) mice. Mouse LPL is virtually undetectable in cKO skeletal and cardiac muscles compared to WT tissue (*p<0.02, **p<0.001; student’s T-test). Error bars represent standard deviation (SD). N = 3 for each genotype. B. Lipoprotein lipase enzyme activity in heart (HRT) and skeletal muscle (SKM) from wild-type (WT) and mck-Cre;lpl -/- (cKO) mice. LPL enzyme activity is significantly decreased in cKO samples compared to WT (p<0.02; Student’s t-test). Error bars represent SD. FFA = free fatty acids. N = 3 for each genotype. C. Average fasting serum triglyceride levels from wild-type (WT) and mck-Cre;lpl -/- (cKO) mice. cKO mice have significantly higher fasting triglycerides levels than WT mice (p<0.0001, Student’s t-test). Error bars represent SD. N = 3 for each genotype.
Fig 2.
RCAS retroviral infection, integration and expression in vitro.
A. An illustration of Mck-Cre-mediated recombination of the Rosa26 locus and LPL conditional allele in mck-Cre;lpl -/-;Rosa26LSLTVA lacZ+/- mice. Cre-mediated recombination deletes LPL and enables Tva expression in striated muscle. Tva expression is required for RCAS retroviral infection, integration and expression in striated muscle. B. (Upper) A diagram of PCR primer binding sites in Rosa26LSLTVA lacZ+/- allele for assessing Cre-mediated recombination efficiency. (Middle) An 800 bp PCR product is present in muscles of mck-Cre;Rosa26LSLTVA lacZ+/- mice but not in liver tissue. The 800 bp PCR product is detectable in hindlimb (HL) and quadriceps (Q) of mck-Cre; Rosa26LSLTVA lacZ+/- mice. A PCR product is not detectable in in liver (L) demonstrating specificity of Cre-mediated recombination. NS denotes a non-specific product at 400 bp. NTC denotes non-template control. (Lower) Representative sequencing alignments of the TVA gene and the sequencing product as well as the non-specific product (NS) obtained from heart gDNA PCR products excised from an agarose gel. LoxP sequences are bold and underlined in each sample. N = 3 for each condition; representative gel image and sequencing alignment shown. C. Active LPL enzyme is secreted from chicken fibroblast cells (DF1) transfected with RCAS plasmid encoding hLPL. No LPL enzyme activity is detected in media from DF1 cells transfected with RCAS plasmid encoding mCherry (p<0.003; Student’s t-test). Error bars represent SD. FFA = free fatty acids. N = 3 for each condition. D. Immunofluorescence images of myofibers isolated from mck-Cre;lpl -/-;Rosa26LSLTVAlacZ+/- mice 72 hours post-infection in vitro with RCAS-hLPL and immunostained for human LPL using an anti-human-specific LPL antibody, 5D2. Human LPL immunoreactivity is present in the myofiber infected with RCAS-hLPL and not detectable in the uninfected control myofiber. Nuclei are counterstained with DAPI (blue) and myofibers are outlined with dotted lines. E. (Upper) A diagram of the restriction fragment length polymorphism used to distinguish between human and mouse LPL mRNA expression in RCAS-hLPL infected myofibers. The Acc1 enzyme digests both the human and mouse RT-PCR products generating unique distinguishable products for each species. (Lower) A ~780 bp band showing amplification of LPL only in myofibers infected with RCAS-hLPL (-Acc1). After digestion (+Acc1) the LPL RT-PCR product is digested into two distinct bands at ~600 bp and ~200 bp, indicative of hLPL not mLPL. No hLPL mRNA expression is detected in myofibers infected with RCAS-mCherry or in white adipose tissue (WAT) from mice infected with RCAS-hLPL retrovirus. NTC denotes non-template control. N = 3 for all conditions; a representative gel image shown.
Fig 3.
Expression of hLPL in striated muscle following in vivo infection with RCAS-hLPL.
A. A schematic of the RCAS rescue strategy and data collection time points. RCAS-hLPL or RCAS-mCherry producing cells were injected into mice at week 0 and 1. Fasting serum triglycerides (TG) were measured at all indicated time points. Post-heparin LPL activity (LPL activity) was measured at baseline, week 2 and week 4 post infection. At 4 weeks post infection, animals were sacrificed, striated muscle samples analyzed for tissue specific LPL activity and myofibers isolated to perform immunostaining for hLPL. B. (Upper) Representative PCR for mCherry gDNA from various tissues of mck-Cre;lpl-/+;Rosa26LSLTVA lacZ+/+ mice two weeks post-injection with RCAS-mCherry DF1 cells or media control. No amplification of mCherry was detected in any tissue two weeks post injection. mCherry plasmid was used as a positive control for amplification. (Lower) Interleukin-2 (IL2) gDNA amplification as a control for gDNA input into original mCherry reaction. NTC = non-template control. n = 3 for each condition. C. Immunofluorescence images of myofibers isolated from mck-Cre;lpl-/-;Rosa26LSLTVA lacZ+/- four weeks post injection with DF1 cells producing RCAS-hLPL retrovirus. Human LPL immunoreactivity is present throughout the myofiber isolated from RCAS-hLPL infected mice and absent from uninjected mice. Nuclei are counterstained with DAPI (blue) and myofibers are outlined with dotted lines. D. Tissue-specific LPL activity in soleus muscle (S) or quadriceps muscle (Q) from mck-Cre;lpl-/-;Rosa26LSLTVA lacZ+/- mice four weeks after injection with RCAS-hLPL DF1 cells (rescue; n = 5) or four weeks post-injection of RCAS-mCherry DF1 cells (control; n = 3). Each symbol represents an individual mouse and bars represent the mean of all samples. E. Tissue specific LPL activity in heart muscle (HRT), white adipose tissue (WAT), or brown adipose tissue (BAT) from mck-Cre;lpl-/-;Rosa26LSLTVA lacZ+/- mice four weeks after injection with RCAS-hLPL DF1 cells (rescue; n = 5) or four weeks after injection with RCAS-mCherry DF1 cells (control; n = 3). WAT and BAT served as positive controls as these tissues produce LPL but do not express MCK. Each symbol represents an individual mouse and bars represent the mean of all samples.
Fig 4.
In vivo RCAS-hLPL infection decreases high triglyceride levels in hypertriglyceridemic mice.
A. A schematic showing the timing measurements for fasting serum triglycerides (TG) or post-heparin LPL activity (PHP LPL) taken prior to (baseline) and following (post-infection) injection of RCAS-hLPL or RCAS-mCherry producing cells. Fasting serum triglycerides (TG) were measured at all indicated time points. B. PHP LPL activity was measured at baseline, week 2 and week 4 post infection. Two weeks post-injection, rescue animal PHP LPL activity is significantly higher than at baseline (p<0.02; One-way ANOVA performed on log transformed values). PHP LPL activity beings to decline by one month post-infection, but remains higher than baseline. PHP LPL activity levels were normalized to baseline levels. Graph shows log transformed values, bars represent the mean of all samples, error bars show +/- SEM. C. Average log transformed fasting serum triglyceride levels from mck-Cre;lpl-/-;Rosa26LSLTVA lacZ+/- mice prior (baseline) and weekly post-injection with RCAS-hLPL DF1 cells (rescue; n = 5) or post-injection with RCAS-mCherry DF1 cells (control; n = 3). At one and three weeks post-infection, rescue mice have significantly lower triglyceride levels compared to baseline (* p<0.05, **p<0.02, One-way ANOVA). By four weeks post injection, rescue triglyceride levels begin to rise, but remain lower than baseline. Triglyceride levels in control mice do not change significantly from baseline following injection of RCAS-mCherry producing cells. Graph shows log transformed values, bars represent the mean of all samples, error bars show +/- SEM.