Cardiac PANK1 deletion exacerbates ventricular dysfunction during pressure overload

Abstract

Coenzyme A (CoA) is an essential cofactor required for intermediary metabolism. Perturbations in homeostasis of CoA have been implicated in various pathologies; however, whether CoA homeostasis is changed and the extent to which CoA levels contribute to ventricular function and remodeling during pressure overload has not been explored. In this study, we sought to assess changes in CoA biosynthetic pathway during pressure overload and determine the impact of limiting CoA on cardiac function. We limited cardiac CoA levels by deleting the rate-limiting enzyme in CoA biosynthesis, pantothenate kinase 1 (Pank1). We found that constitutive, cardiomyocyte-specific Pank1 deletion (cmPank1^-/-) significantly reduced PANK1 mRNA, PANK1 protein, and CoA levels compared with Pank1-sufficient littermates (cmPank1^+/+) but exerted no obvious deleterious impact on the mice at baseline. We then subjected both groups of mice to pressure overload-induced heart failure. Interestingly, there was more ventricular dilation in cmPank1^-/- during the pressure overload. To explore potential mechanisms contributing to this phenotype, we performed transcriptomic profiling, which suggested a role for Pank1 in regulating fibrotic and metabolic processes during the pressure overload. Indeed, Pank1 deletion exacerbated cardiac fibrosis following pressure overload. Because we were interested in the possibility of early metabolic impacts in response to pressure overload, we performed untargeted metabolomics, which indicated significant changes to metabolites involved in fatty acid and ketone metabolism, among other pathways. Collectively, our study underscores the role of elevated CoA levels in supporting fatty acid and ketone body oxidation, which may be more important than CoA-driven, enzyme-independent acetylation in the failing heart.NEW & NOTEWORTHY Changes in CoA homeostasis have been implicated in a variety of metabolic diseases; however, the extent to which changes in CoA homeostasis impacts remodeling has not been explored. We show that limiting cardiac CoA levels via PANK deletion exacerbated ventricular remodeling during pressure overload. Our results suggest that metabolic alterations, rather than structural alterations, associated with Pank1 deletion may underlie the exacerbated cardiac phenotype during pressure overload.

Keywords: CoA metabolism; Pank1; fibrosis; heart failure; pantothenate kinase 1.

Figure 1.

Cardiomyocyte-specific pantothenate kinase 1 (Pank1) deletion limits cardiac mitochondria acetylation. We characterized the impact of cardiomyocyte Pank1 deletion on cardiac mitochondrial acetylation. A: cardiac Pank mRNA expression. B: cardiac PANK1 protein expression. C: coenzyme A (CoA) levels in the heart and skeletal muscle. D: mitochondria acetylation levels via immunoblot. E: quantification of acetylation levels at indicated molecular weights. An unpaired Student’s t test was used to determine significant differences between cmPank1^+/+ and cmPank1^−/− groups. cmPank1^−/−, cardiomyocyte-specific Pank1-deficient mice.

Figure 2.

Cardiomyocyte-specific pantothenate kinase 1 (Pank1) deletion does not affect ventricular dilation, fibrosis, and vascular density in naïve mice hearts. A: end-diastolic function (EDV). B: end-systolic function (ESV). C: ejection fraction (EF). D: trichrome-stained hearts. E: quantification of fibrosis in trichrome-stained hearts. F: isolectin B4-stained (green) hearts to determine vascular density. G: quantification of vascular density in isolectin B4-stained hearts. An unpaired Student’s t test was used to determine significant differences between cmPank1^+/+ and cmPank1^−/− groups. cmPank1^−/−, cardiomyocyte-specific Pank1-deficient mice.

Figure 3.

Cardiomyocyte-specific pantothenate kinase 1 (Pank1) deletion does not affect cardiomyocyte size in naïve mice hearts. We assessed the impact of Pank1 deletion on cardiomyocyte size by wheat germ agglutinin (WGA) and measuring heart weights. A: WGA-stained (red) cmPank1^+/+ and cmPank1^−/− hearts. B: quantification of WGA-stained hearts. C: heart weight/tibia length ratio. An unpaired Student’s t test was used to determine significant differences between cmPank1^+/+ and cmPank1^−/− groups. cmPank1^−/−, cardiomyocyte-specific Pank1-deficient mice; CSA, cross-sectional area; HW, heart weight.

Figure 4.

Pantothenate kinase 1 (Pank1) deletion exacerbates ventricular dilation during pressure overload. Mice were subjected to transverse aortic constriction (TAC), and echocardiography was performed at 2, 4, 8, and 12 wk after TAC. A: left ventricular end-diastolic volumes (EDV). B: left ventricular end-systolic volumes (ESV). C: ejection fraction (EF). An unpaired Student’s t test was used to determine significant differences between cmPank1^+/+ and cmPank1^−/− groups at each timepoint. cmPank1^−/−, cardiomyocyte-specific Pank1-deficient mice.

Figure 5.

Cardiomyocyte pantothenate kinase 1 (Pank1) deletion impacts transcriptional profile of fibrotic and metabolic processes at 12-wk postpressure overload. Total RNA was isolated from hearts 12-wk post-TAC, and RNA-seq was performed. A: volcano plot of differentially expressed genes. B: heat map showing downregulated genes in male (blue) and female (purple) hearts. C: heat map showing upregulated genes in male (blue) and female (purple) hearts. D: Gene Ontology (GO) enrichment analysis showing biological processes associated with downregulated and upregulated genes in cmPank1^−/− compared with cmPank1^+/+ hearts. cmPank1^−/−, cardiomyocyte-specific Pank1-deficient mice; TAC, transverse aortic constriction.

Figure 6.

Cardiomyocyte pantothenate kinase 1 (Pank1) deletion increases cardiac fibrosis 12-wk post-TAC. A: trichrome-stained cmPank1^+/+ and cmPank1^−/− hearts 12-wk post-TAC. B: quantification of fibrosis in trichrome-stained hearts 12-wk post-TAC. RNA-seq data showing cardiac Acta2 mRNA expression 12-wk post-TAC (C), cardiac Gdf6 mRNA expression 12-wk post-TAC (D), cardiac Fbn2 mRNA expression 12-wk post-TAC (E), cardiac Tnc mRNA expression 12-wk post-TAC (F). An unpaired Student’s t test was used to determine significant differences between cmPank1^+/+ and cmPank1^−/− groups. cmPank1^−/−, cardiomyocyte-specific Pank1-deficient mice; Fbn2, fibrilin-2; Gdf6, growth differentiation factor 6; TAC, transverse aortic constriction; Tnc, tenascin C.

Figure 7.

Cardiomyocyte pantothenate kinase 1 (Pank1) deletion alters metabolites involved in substrate utilization after 2 wk of pressure overload. A: principal component analysis (PCA) plot. B: metabolite set enrichment analysis in naïve animals. C: variable of importance (VIP) plot of animals subjected to TAC. D: metabolite set enrichment analysis in animals subjected to TAC. cmPank1^−/−, cardiomyocyte-specific Pank1-deficient mice; TAC, transverse aortic constriction.