AAV-mediated overexpression of CPT1B protects from cardiac hypertrophy and heart failure in a murine pressure overload model

Fig. 1. CPT1B mRNA level is lower in various models of heart insufficiency. a Statistical quantification of CPT1B mRNA levels in NRVCMs after treatment with phenylephrine (PE), endothelin-1 (ET-1) or isoprenaline. Values were normalized to untreated cardiomyocytes as a control. n = 6 b Statistical quantification of CPT1B mRNA levels in MLP knock-out (MLP-KO) compared to, normalized to wild-type littermates, n = 3. c Statistical quantification of CPT1B mRNA levels in mice after transverse aortic constriction (TAC) vs. sham-operated mice, normalized to sham mice. n = 8. d Statistical quantification of CPT1B mRNA levels in human myocardial biopsies from non-failing controls and patients with hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM) and idiopathic cardiomyopathy (ICM). GAPDH (a-c) and RPL32 (d) were used as a housekeeping gene, n = 3 for control (non-failing, NF) and n = 10 for patient samples. (*p < 0.05, **p<0.01, ***p < 0.001, quantitative data are presented as means ± SD)

Fig. 2. AAV6-mediated overexpression of CPT1B ameliorates PE-induced cardiomyocyte hypertrophy in NRVCMs. a Statistical quantification of CPT1B mRNA levels in NRVCMs after transduction with AAV6. b Representative images showing CPT1B immunohistochemistry (green). Mitochondria were labeled with the mitotracker dye (red), and nuclei were stained with DAPI (blue); scale bar = 25 µm. c Statistical quantification of mean green fluorescence intensity in transduced cells. Sixteen images were analyzed/condition. d Statistical quantification of cardiomyocyte cross-sectional area, measured following staining with α-actinin 2 as a cytoskeleton protein. e, f Quantification of fetal gene program in NRVCMs in the depicted treatment groups. (**p < 0.01, ***p < 0.001, one-way ANOVA, quantitative data are presented as means ± SD, n = 4)

Fig. 3. AAV6-mediated overexpression of CPT1B ameliorates PE-induced mitochondrial dysfunction. a Illustrative images showing MitoSOX staining (red) of NRVCMs subjected to the depicted treatments. b Statistical quantification of relative red fluorescent signal measured by ImageJ. Cells transduced with AAV6-Luc served as controls. c Representative confocal images of NRVCMs treated with TMRE as an indicator of mitochondrial membrane potential and d quantification of relative mean red fluorescence intensity; scale bar = 25 µm. (**p < 0.01, ***p < 0.001, one-way ANOVA, quantitative data are presented as means ± SD, n = 4, 16 images analyzed/condition)

Fig. 4. CPT1B overexpression induces palmitate oxidation in NRVCMs. a Maximal respiration rate, as a measure of palmitate oxidation in the depicted experimental groups. BSA: bovine serum albumin, PalmBSA: saturated fatty acid complex composed of 6:6 palmitate:BSA, Eto: etomoxir. b Representative diagram showing oxygen consumption rate (OCR) acquired during glycolysis stress test assay. c, d, e Assessment of glycolysis, glycolytic reserve and glycolytic capacity following CPT1B overexpression in NRVCMs, measured by Seahorse assay. f Representative images showing BODIPY staining in NRVCMs subjected to AAV6-CPT1B and AAV6-Luc transduction and g statistical quantification of mean fluorescence intensity, as a marker of lipid droplets accumulation in cardiac myocytes (***p < 0.001, one-way ANOVA, quantitative data are presented as means ± SD, n = 3)

Fig. 5. AAV9-mediated overexpression of CPT1B in cardiomyocytes prevents deterioration of cardiac function. a Graphical representation of the experimental timeline (w = weeks). b Echocardiographic measurement of ejection fraction over the four time points (baseline; 2, 4, and 6 weeks after TAC). (c) Echocardiographic measurement of fractional shortening over the four time points. d Echocardiographic measurement of the left ventricular end-diastolic diameter over the four time points. e Statistical quantification of CPT1B mRNA levels in cardiac tissue of mice subjected to the depicted treatments. Values were normalized to GAPDH, which served as housekeeping gene and expressed as fold-change over the sham group. f Illustrative confocal images demonstrating efficient CPT1B expression in cardiomyocytes after systemic injection of AAV9-CPT1B; scale bar = 100 µm. g Statistical quantification of CPT1B protein level in the myocardium of mice subjected to the treatments shown. (n = 8, comparison sham group vs. AAV9-Luc-treated TAC-group: *p < 0.05, **p < 0.01, ***p < 0.001, comparison AAV9-treated TAC-group vs. AAV9-CPT1B treated TAC-group: #p < 0.05, ##p < 0.01, ###p < 0.001, one-way ANOVA,…

Fig. 6. AAV9-mediated overexpression of CPT1B attenuates cardiac hypertrophy and extracellular matrix deposition. a Quantification of Nppa, Nppb, and Myh7 mRNA levels as markers of a pro-hypertrophic response in mice subjected to the treatments shown. Values were normalized to GAPDH, which served as housekeeping gene and expressed as fold-change over the sham group. b Quantification of Col1a1 and Col3a1 mRNA levels as markers of cardiac fibrosis in mice subjected to the treatments shown. Values were normalized to GAPDH, which served as housekeeping gene and expressed as fold-change over the sham group. c Illustrative images showing heart sections with Masson’s trichrome staining to illustrate pathological extracellular matrix deposition of groups as indicated (scale bar = 100 µm) and d statistical quantification of the percentage of blue area marking pathological extracellular matrix deposition (40 images analyzed per group). Values were expressed as fold-changed over the sham group. e Illustrative images of heart sections stained with WGA as a membrane marker (red) on which cell size measurements shown in Fig. 3f were performed; scale bar = 100 µm. f Statistical quantification of…