Carnitine palmitoyltransferase-1b deficiency aggravates pressure overload-induced cardiac hypertrophy caused by lipotoxicity

Abstract

Background: Carnitine palmitoyltransferase-1 (CPT1) is a rate-limiting step of mitochondrial β-oxidation by controlling the mitochondrial uptake of long-chain acyl-CoAs. The muscle isoform, CPT1b, is the predominant isoform expressed in the heart. It has been suggested that inhibiting CPT1 activity by specific CPT1 inhibitors exerts protective effects against cardiac hypertrophy and heart failure. However, clinical and animal studies have shown mixed results, thereby creating concerns about the safety of this class of drugs. Preclinical studies using genetically modified animal models should provide a better understanding of targeting CPT1 to evaluate it as a safe and effective therapeutic approach.

Methods and results: Heterozygous CPT1b knockout (CPT1b(+/-)) mice were subjected to transverse aorta constriction-induced pressure overload. These mice showed overtly normal cardiac structure/function under the basal condition. Under a severe pressure-overload condition induced by 2 weeks of transverse aorta constriction, CPT1b(+/-) mice were susceptible to premature death with congestive heart failure. Under a milder pressure-overload condition, CPT1b(+/-) mice exhibited exacerbated cardiac hypertrophy and remodeling compared with wild-type littermates. There were more pronounced impairments of cardiac contraction with greater eccentric cardiac hypertrophy in CPT1b(+/-) mice than in control mice. Moreover, the CPT1b(+/-) heart exhibited exacerbated mitochondrial abnormalities and myocardial lipid accumulation with elevated triglycerides and ceramide content, leading to greater cardiomyocyte apoptosis.

Conclusions: CPT1b deficiency can cause lipotoxicity in the heart under pathological stress, leading to exacerbation of cardiac pathology. Therefore, caution should be exercised in the clinical use of CPT1 inhibitors.

Figure 1

CPT1b mRNA level and activity in CPT1b^+/− mice. Mice were sacrificed at 12~14 weeks of age. RNA samples were extracted from ventricular tissues. A & C) Transcript level of CPT1b and CPT1a were determined by Q-PCR, results from each gene/primer pair were normalized to β-actin (n=4). B) Protein expression was determined by western blot (n=4). D) CPT1 activity was measured using isolated mitochondria according to the method described in the on-line supplement (n=4). E & F) Palmitate oxidation rate and glucose oxidation rate were measured in isolated working heart (n=6). *p<0.05 vs WT.

Figure 2

Echocardiographic parameters of the mice with pressure-overload. Mice were subjected to TAC procedures at 10~12 weeks of age. A). Kaplan-Meier survival curves for WT and CPT1b^+/− mice that were subjected to severe TAC-induced pressure-overload. The survival curves were statistically different (P < 0.05) by log-rank test. B, C, D & E). Mice were subjected to modest TAC at 10~12 weeks of age. Representative echocardiographic images of M-mode measurement and echocardiographic results of EF, FS, and corrected left ventricular mass to body weight (2 weeks after TAC, *p<0.05 vs sham, ^#P<0.05 vs WT TAC. Sham: n=9; TAC: n=11).

Figure 3

Transcript levels of molecular markers of pathological cardiac hypertrophy in CPT1b+/− mice with pressure-overload. Mice were subjected to a modest pressure-overload condition at 10~12 weeks of age and sacrificed two weeks after TAC. A & B). Heart (mg)/ body weight (g) ratio and heart weight (mg) to tibia length (mm), *p<0.05 vs sham (n=6), # p<0.05 vs WT TAC (n=13). C). Real-time PCR assessment of natriuretic peptide precursor A (Nppa), B (Nppb) and myosin heavy chain-β (MHC-β) transcripts, *p<0.05 vs sham, # p<0.05 vs WT TAC, (n=5).

Figure 4

Cardiac histology and ultrastructure in CPT1b^+/− mice with pressure-overload. Mice were subjected to TAC procedures at 10~12 weeks of age and sacrificed two weeks after TAC. A) Representative histological images (400 ×) with H&E staining on heart section. B) Relative cross-sectional areas. The mean cardiomyocyte cross-sectional area in sham WT mice was set as 1. *p<0.05 vs sham. ^#p<0.05 vs WT TAC (n=6). C) Representative images of heart sections stained with Trichrome blue. D) Relative fibrosis areas. The whole section area was set as 100%. E) Representative images of left ventricular TEM assessment (×1100). F) Quantification results of mitochondrial volume (%) of heart sections from TEM images. *p<0.05 vs sham. ^#p<0.05 vs WT TAC (n=8).

Figure 4

Cardiac histology and ultrastructure in CPT1b^+/− mice with pressure-overload. Mice were subjected to TAC procedures at 10~12 weeks of age and sacrificed two weeks after TAC. A) Representative histological images (400 ×) with H&E staining on heart section. B) Relative cross-sectional areas. The mean cardiomyocyte cross-sectional area in sham WT mice was set as 1. *p<0.05 vs sham. ^#p<0.05 vs WT TAC (n=6). C) Representative images of heart sections stained with Trichrome blue. D) Relative fibrosis areas. The whole section area was set as 100%. E) Representative images of left ventricular TEM assessment (×1100). F) Quantification results of mitochondrial volume (%) of heart sections from TEM images. *p<0.05 vs sham. ^#p<0.05 vs WT TAC (n=8).

Figure 4

Cardiac histology and ultrastructure in CPT1b^+/− mice with pressure-overload. Mice were subjected to TAC procedures at 10~12 weeks of age and sacrificed two weeks after TAC. A) Representative histological images (400 ×) with H&E staining on heart section. B) Relative cross-sectional areas. The mean cardiomyocyte cross-sectional area in sham WT mice was set as 1. *p<0.05 vs sham. ^#p<0.05 vs WT TAC (n=6). C) Representative images of heart sections stained with Trichrome blue. D) Relative fibrosis areas. The whole section area was set as 100%. E) Representative images of left ventricular TEM assessment (×1100). F) Quantification results of mitochondrial volume (%) of heart sections from TEM images. *p<0.05 vs sham. ^#p<0.05 vs WT TAC (n=8).

Figure 5

Apoptosis assay of CPT1b^+/− mice with pressure-overload. Mice were subjected to TAC procedures at 10~12 weeks of age and sacrificed two weeks after TAC. A) Representative image of apoptotic cardiomyocytes in a section of mice hearts. Green staining (see white arrows) indicates apoptotic cells, blue indicates nuclei (TUNEL fluorescence FITC kit, GenScript,USA). B) Quantification results of TUNEL assay (n=5). C) Western blots of cytochrome c in cytosolic and mitochondrial fractions extracted from hearts of mice. *p<0.05 vs sham. ^#p<0.05 vs WT TAC (n=4).

Figure 5

Apoptosis assay of CPT1b^+/− mice with pressure-overload. Mice were subjected to TAC procedures at 10~12 weeks of age and sacrificed two weeks after TAC. A) Representative image of apoptotic cardiomyocytes in a section of mice hearts. Green staining (see white arrows) indicates apoptotic cells, blue indicates nuclei (TUNEL fluorescence FITC kit, GenScript,USA). B) Quantification results of TUNEL assay (n=5). C) Western blots of cytochrome c in cytosolic and mitochondrial fractions extracted from hearts of mice. *p<0.05 vs sham. ^#p<0.05 vs WT TAC (n=4).

Figure 6

Myocardial lipid accumulation of mice with pressure-overload. Mice were subjected to TAC procedures at 10~12 weeks of age and sacrificed two weeks after TAC. A) Myocardial triglyceride content in hearts (n=5). B) Representative photomicrographs depicting the histologic appearance of ventricular tissue sections stained with Oil red O. C) Ceramide species were quantified by ESI-MS/MS as described in the methods (on line supplement). D) Total ceramide was calculated from the sum of C16:0, C18:0, C20:0, C22:0, and C24:0 ceramide subspecies. *p<0.05 vs sham. ^#p<0.05 vs WT TAC (n=4).

Figure 7

The expression of fatty acid uptake proteins and mitochondrial biogenesis in mice subjected to pressure-overload. Mice were subjected to TAC procedures at 10~12 weeks of age and sacrificed two weeks after TAC. A) QPCR assessment of CD36, FABP and FATP transcripts (n=4). B) Western blot images and quantification of CD36 and FATP (n=4). C) QPCR assessment of mitochondrial biogenesis genes transcripts (n=4). D) Western blots image and quantification of PGC1-α and TFAM (n=4). E) Mitochondia DNA copy number was assessed by ratio of mitochondrial gene (Cyto b) and nuclear gene (Rcan-1) as described in the methods (on line supplement). *p<0.05 vs sham. ^#p<0.05 vs WT TAC (n=6).

Figure 7

The expression of fatty acid uptake proteins and mitochondrial biogenesis in mice subjected to pressure-overload. Mice were subjected to TAC procedures at 10~12 weeks of age and sacrificed two weeks after TAC. A) QPCR assessment of CD36, FABP and FATP transcripts (n=4). B) Western blot images and quantification of CD36 and FATP (n=4). C) QPCR assessment of mitochondrial biogenesis genes transcripts (n=4). D) Western blots image and quantification of PGC1-α and TFAM (n=4). E) Mitochondia DNA copy number was assessed by ratio of mitochondrial gene (Cyto b) and nuclear gene (Rcan-1) as described in the methods (on line supplement). *p<0.05 vs sham. ^#p<0.05 vs WT TAC (n=6).

Figure 7

The expression of fatty acid uptake proteins and mitochondrial biogenesis in mice subjected to pressure-overload. Mice were subjected to TAC procedures at 10~12 weeks of age and sacrificed two weeks after TAC. A) QPCR assessment of CD36, FABP and FATP transcripts (n=4). B) Western blot images and quantification of CD36 and FATP (n=4). C) QPCR assessment of mitochondrial biogenesis genes transcripts (n=4). D) Western blots image and quantification of PGC1-α and TFAM (n=4). E) Mitochondia DNA copy number was assessed by ratio of mitochondrial gene (Cyto b) and nuclear gene (Rcan-1) as described in the methods (on line supplement). *p<0.05 vs sham. ^#p<0.05 vs WT TAC (n=6).

Figure 8

Fatty acid and glucose oxidation rates, and CPT1 activity/expression in pressure-overloaded hearts. Mice were subjected to TAC procedures at 10~12 weeks of age and sacrificed 3 days, 1week and two weeks after TAC, respectively. A) Fatty acid oxidation rate was determined on isolated mitochondria (n=6). B) Glucose oxidation rate was determined on heart homogenate (n=6). C) CPT1 activity was assayed using isolated mitochondria (n=6). D) QPCR assessment of CPT1b, 2 weeks after TAC (n=4). E) Western blots image and quantification of CPT1b, 2 weeks after TAC. *p<0.05 vs sham. ^#p<0.05 vs WT TAC. ^Δp<0.05 vs WT Sham (n=6).

Figure 8

Fatty acid and glucose oxidation rates, and CPT1 activity/expression in pressure-overloaded hearts. Mice were subjected to TAC procedures at 10~12 weeks of age and sacrificed 3 days, 1week and two weeks after TAC, respectively. A) Fatty acid oxidation rate was determined on isolated mitochondria (n=6). B) Glucose oxidation rate was determined on heart homogenate (n=6). C) CPT1 activity was assayed using isolated mitochondria (n=6). D) QPCR assessment of CPT1b, 2 weeks after TAC (n=4). E) Western blots image and quantification of CPT1b, 2 weeks after TAC. *p<0.05 vs sham. ^#p<0.05 vs WT TAC. ^Δp<0.05 vs WT Sham (n=6).