Cardiomyocyte expression of PPARγ leads to cardiac dysfunction in mice (original) (raw)
Creation of cardiac-specific PPARγ1–transgenic mice. Mice overexpressing PPARγ in cardiomyocytes via the α-MHC promoter were designated MHC-PPARγ1 mice (Figure 1A). PCR screening of the offspring identified 3 founders harboring the MHC-PPARγ1 transgene. Offspring from 2 of these founders, designated MHC-PPARγ1L (low-expressing) and MHC-PPARγ1H (high-expressing), were used in this report. Male mice were used in the experiments unless indicated otherwise. The PPARγ1 transgene was expressed specifically in the heart; no expression was detected in the liver, skeletal muscle, or fat (Figure 1B). In control mouse hearts, PPARγ1 mRNA levels were low; the ratio of PPARγ to 18S rRNA was much lower in control heart than adipose tissue. In contrast, MHC-PPARγ1L mice had PPARγ to 18S rRNA levels approximately twice those found in adipose tissue (Figure 1C). MHC-PPARγ1H hearts had PPARγ mRNA levels approximately 10-fold higher than those in MHC-PPARγ1L hearts. PPARγ protein levels were 1.9-fold (MHC-PPARγ1L) and 7.2-fold (MHC-PPARγ1H) higher in the cardiac ventricles of transgenic mice than littermate controls (Figure 1, D and E).
Construct and gene expression in PPARγ1-transgenic mice. (A) Diagram of PPARγ1 genomic locus and PPARγ1 construct design. The α-MHC promoter was used to drive PPARγ1 cDNA expression. Black boxes indicate exons that are numbered. PCR primers are indicated. Boxes A1 and A2 indicate PPARγ1-specific exons. (B) Cardiac-specific MHC-PPARγ1 expression. RT-PCR analysis of RNA was performed in 3-month-old MHC-PPARγ1L male mice using primer A: 5ι end specific to α-MHC promoter exon 2 and 3′ end specific to PPARγ1 cDNA nucleotides 173–192. (C) Heart expression of total PPARγ mRNA (both endogenous gene and transgene) in MHC-PPARγ1L and MHC-PPARγ1H male mice was quantified by qRT-PCR. Primer C was used for PCR amplification. Data are shown as mean ratio (±SD; n = 5 in each group) corrected for 18S rRNA. ***P < 0.001 for MHC-PPARγ1 mice versus control mice. (D) Nuclear protein (30 μg) from heart tissues of 4- and 8-month-old MHC-PPARγ1L and 4-month-old MHC-PPARγ1H mice and their littermate controls was analyzed by Western blot using polyclonal PPARγ antibody. Western blot for lamin B1 is shown as a control. (E) Bands were quantified using Molecular Analysis Software. Data were normalized to values for littermate controls (set at 1.0), and normalized units of expression are shown (±SD; n = 3 in each group). **P < 0.01; ***P < 0.001 for MHC-PPARγ1 versus control mice. C, littermate control; Tg, transgenic MHC-PPARγ1 mouse; PA, poly(A) site.
Transgenic mice from each of the lines bred normally, and glucose, body weight, and plasma lipid levels were similar to those of nontransgenic mice (Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI30335DS1).
PPARγ activity in hearts of MHC-PPARγ1 mice. We estimated the degree of PPARγ activity in the MHC-PPARγ1 mice by assessing expression of downstream genes. Surprisingly, despite the adipose-like PPARγ1 expression level, hearts from young MHC-PPARγ1L mice (4 months old) had an expression of downstream genes similar to that of controls (Figure 2A). Eight-month-old MHC-PPARγ1L mice, however, had greater expression of acyl-CoA oxidase (AOX) (1.35-fold), carnitine palmitoyl transferase–1 (CPT1) (1.63-fold), FA translocase (CD36) (1.61-fold), FA synthase (FAS) (1.44-fold), and adipose differentiation–related protein (ADRP) (1.81-fold) (Figure 2B).
Expression of PPARγ target genes in the hearts of MHC-PPARγ1 mice. (A and B) Four- and 8-month-old MHC-PPARγ1L male mice; (C) 4-month-old PPARγ1H male mice. Data are shown as mean ratio (±SD; n = 5–9 in each group) corrected for 18S rRNA and normalized to those for littermate controls (set as 1.0). *P < 0.05, **P < 0.01, ***P < 0.001 for MHC-PPARγ1 mice versus littermate controls.
In contrast, multiple genes were markedly upregulated in the hearts of 4-month-old MHC-PPARγ1H mice (Figure 2C), including genes involved in lipid oxidation, uptake, synthesis, and storage, such as AOX, CPT1, CD36, FAS, ADRP, and SREBP1. Expression of ATP-binding cassette transport A1 (ABCA1), which mediates reverse cholesterol transport to apoA-I and is induced by PPARγ (18), was 1.73-fold higher in 4-month-old MHC-PPARγ1 H mice (Figure 2C).
PPAR gene expression in control and MHC-PPARγ mice with aging. To determine why the MHC-PPARγL transgene did not alter downstream gene expression in the young mice, we assessed expression of PPAR family members in control and transgenic mice with aging. As noted by others (19), PPARα expression decreased as the mice aged (Supplemental Figure 1A). Moreover, expression of PPARα was reduced by 29% and 60% in the 2- and 9-month-old MHC-PPARγL transgenic mice, respectively, compared with their littermate controls. Neither endogenous and transgenic PPARγ1 nor PPARδ expression differed between 2- and 9-month-old mouse hearts (Supplemental Figure 1, B and C). Thus, changes in PPARα expression might modify the downstream effects of the MHC-PPARγL transgene.
Cardiac lipid content and uptake in MHC-PPARγ1 mice. Greater CD36 expression in the MHC-PPARγ1 mice could increase uptake of plasma free and lipoprotein-derived FA. Heart TG and FA content were increased in 8-month-old MHC-PPARγ1L mice (Figure 3A). Oil red O staining revealed greater accumulation of intracellular neutral lipids in hearts from MHC-PPARγ1L mice compared with littermate controls (Figure 3, B and C). Electron microscopy showed more lipid droplets within the sarcoplasm of cardiomyocytes, with distortion of the mitochondrial contours in MHC-PPARγ1L mice (Figure 3D). In some areas the cristae were disrupted (Figure 3E).
Increased heart lipid accumulation in MHC-PPARγ1 mice. (A) Heart TG (left panel) and FFA content (right panel) was significantly increased in MHC-PPARγ1L transgenic mice (n = 7 for each group). (B) Oil red O staining showed an abundance of neutral lipid droplets randomly scattered throughout the cytoplasm of cardiomyocytes in MHC-PPARγ1L female mice (right panel) after 24-hour fasting (original magnification, ×400). (C) Oil red O staining was quantified using Molecular Analysis Software (n = 3 in each group). Data were normalized to values for littermate controls (set as 1.0). (D). Electron micrographs (original magnification, ×15,000) of myocardial tissue showed a large increase in lipid droplets within the sarcoplasm of cardiomyocytes in MHC-PPARγ1L male mice (right panel) compared with littermates (left panel). All of these lipid droplets were located adjacent to mitochondria, with distortion of the mitochondrial contours. (E) Electron micrographs (original magnification, ×50,000) detailed distorted architecture of the mitochondrial inner matrix with electron lucent foci (arrows) in MHC-PPARγ1L mice, and in some areas the cristae were disrupted. (F) [14C-TG]VLDL uptake into heart of MHC-PPARγ1L male mice and littermate control mice at 8 months of age (n = 6 per group). LD, lipid droplet; M, mitochondria. Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared with littermate controls. DPM, decays per minute.
Cardiac uptake of VLDL-TG in 8-month-old MHC-PPARγ1L hearts was 74% greater than that in littermate control hearts (P < 0.01; Figure 3F), which corresponds with increased expression of lipid uptake genes and cardiac lipid storage. Liver and skeletal muscle lipid uptake were not altered by the PPARγ1 transgene (data not shown).
Glucose transporter 4 expression and glucose uptake in MHC-PPARγ1 mice. Unlike in MHC-PPARα transgenic mice (8), glucose transporter 4 (GLUT4) and GLUT1 mRNA levels were unchanged in the MHC-PPARγ1L mice (data not shown) and were upregulated in MHC-PPARγ1H mice (Table 1). Pyruvate dehydrogenase kinase 4, the gene regulating glucose oxidation, was also downregulated in the MHC-PPARγ1H mice (Table 1).
Expression of glucose metabolism and GLUT4 regulation genes in 4-month-old MHC-PPARγ1H mice
The GLUT4 gene undergoes a complex program of gene regulation. The GLUT4–myocyte enhancer factor 2 (GLUT4-MEF2) site is required for metabolic regulation of GLUT4 transcription (20–22). MEF2A, MEF2C, and MEF2D isoforms, but not MEF2B isoforms, are expressed in the heart. PPARγ coactivator 1 (PGC1) also interacts with MEF2C to upregulate endogenous GLUT4 expression and glucose uptake in cultured muscle cells (23). GLUT4 expression in MHC-PPARγ1H mice was associated with increased expression of MEF2C and MEF2D (1.48-fold, P < 0.001, and 1.55-fold, P = 0.009, respectively), but with no change in MEF2A. PGC1β gene expression was also upregulated (1.68-fold; P = 0.01) in the hearts of the MHC-PPARγ1H mice (Table 1).
Myocardial glucose import in vivo was assessed using 2-deoxy-d-[3H]glucose. Heart uptake of glucose was unchanged in the MHC-PPARγ1L hearts (Figure 4A, left panel) and was increased by 37% in MHC-PPARγ1H mice compared with littermate controls (P < 0.05; Figure 4A, right panel). The PAS technique revealed greater cardiac glycogen storage in MHC-PPARγ1L mice compared with littermate controls (Figure 4B). PAS diastase (PAS-D) completely removed the glycogen in cardiac sections (Figure 4C). These data indicate that in the MHC-PPARγ mice, although cardiac lipid uptake was elevated, glucose uptake was not reduced.
Increased cardiac glucose uptake and glycogen storage in MHC-PPARγ1 male mice. (A) 2-Deoxy-d-[3H]glucose uptake into heart of MHC-PPARγ1 and wild-type control mice. MHC-PPARγ1L mice were 8 months old (n = 7 in each group); MHC-PPARγ1H mice were 4 months old (n = 6 in each group). (B) PAS staining of heart tissue from 8-month-old MHC-PPARγ1L female mice (middle panel) and littermate controls (left panel) after 6-hour fasting (original magnification, ×400). The arrow shows focal increased staining. Glycogen content was quantified using Molecular Analysis software (n = 3–4 per group) and normalized to values for PAS-staining controls (set as 1.0) (right panel). G, glycogen. Data are shown as mean ± SD. *P < 0.05 compared with littermate controls; **P < 0.01. (C) The glycogen staining was removed by PAS diastase (PAS-D) digestion (left and middle panels), and the data were quantified using Molecular Analysis software (right panel).
Cardiac function in MHC-PPARγ1 mice. Both MHC-PPARγ1L and MHC-PPARγ1H hearts had dilated left ventricles and impaired systolic function. Heart to body weight ratios were increased in 8-month-old MHC-PPARγ1L and 4-month-old MHC-PPARγ1H mice (Figure 5, A and B). The dilated cardiomyopathy phenotype was grossly visible (Figure 5, C and D) and also seen by echocardiography (Figure 5, E–J). Left ventricular systolic dimension was increased (0.17 ± 0.03 versus 0.14 ± 0.01 cm; P = 0.021), and fractional shortening was reduced from 53% ± 3.6% to 48% ± 5.2% (P = 0.036) by age 8 months in PPARγ1L mice (Figure 5, F and G). The MHC-PPARγ1H mice had more severe cardiac dysfunction. Four-month-old PPARγ1H hearts had greater left ventricular systolic dimension (0.28 ± 0.02 versus 0.17 ± 0.03; P < 0.001) and a greater reduction in fractional shortening (from 47.3% ± 5.3% to 30.2% ± 3.2%; P < 0.001) (Figure 5, I and J).
Dilated cardiomyopathy in MHC-PPARγ1 mice. (A and B) The heart to body weight ratio was increased in both MHC-PPARγ1L (n = 11–13) and MHC-PPARγ1H male mice (n = 8–9). (C) Representative photographs of hearts of control and MHC-PPARγ1L male mice. (D) Histological section of H&E-stained cardiac tissue in control and MHC-PPARγ1L male mice. (E and H) Representative echocardiographic images of left ventricle motion in MHC-PPARγ1L and MHC-PPARγ1H mice. Echocardiography showed increased left ventricular systolic dimension (F and I) and reduced fractional shortening (G and J) in both MHC-PPARγ1L and MHC-PPARγ1H mice. FS, fractional shortening; LVDs, left ventricular end-systolic dimension. Data are shown as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 versus littermate controls.
Expression of heart failure marker genes brain-type natriuretic peptide (BNP) and atrial natriuretic factor (ANF), was increased in MHC-PPARγ1 mice (Table 2). These increases were seen by 4 months in the PPARγ1H line but not until 8 months in the PPARγ1L line.
Expression of heart failure marker genes in MHC-PPARγ1L and MHC-PPARγ1H mice
Effects of rosiglitazone in wild-type and MHC-PPARγ1 mice. In contrast to other lipotoxic heart models (13, 16, 24), rosiglitazone treatment of 8-month-old MHC-PPARγ1L mice led to further deterioration of cardiac function: increased lipid accumulation, larger hearts, and decreased fractional shortening (Figure 6, A and B). This was associated with increased cardiac gene expression of CD36 and ADRP in rosiglitazone-treated MHC-PPARγ1L mice (Figure 6C). When wild-type mice were treated with rosiglitazone, cardiac expression of usual PPARγ downstream genes was paradoxically reduced in the 8-month-old mice (Figure 6C). Adipose tissue and muscle had increased CD36 expression after rosiglitazone treatment (Supplemental Figure 2).
MHC-PPARγ1L and C57BL/6 wild-type mice with or without rosiglitazone treatment. (A) Oil red O heart histology of 8-month-old MHC-PPARγ1L female mice with (middle panel) or without (left panel) rosiglitazone treatment. Staining was quantified and normalized to values for littermate controls (set as 1.0) (right panel). Data are shown as mean (± SD; n = 3–4 in each group). (B) Heart to body weight ratio (left panel) and echocardiographic measurements of fractional shortening (right panel) in 8-month-old MHC-PPARγ1L male mice with or without rosiglitazone treatment. (C) qRT-PCR analysis of cardiac gene expression in 8-month-old male C57BL/6 wild-type (left panel) and MHC-PPARγ1L mice (right panel) with or without rosiglitazone treatment (n = 6–7 per group). *P < 0.05, **P < 0.01, and ***P < 0.001 versus the group without rosiglitazone treatment group. Rosi, rosiglitazone, WT, wild-type C57BL/6 mice.
Pathways leading to cardiac dysfunction in MHC-PPARγ1 mice. To explore potential mechanisms in the development of lipid-induced cardiomyopathy, we examined heart tissue for evidence of lipid accumulation triggering programmed cell death. Four-month-old MHC-PPARγ1H mice had a 40% increase in levels of cardiac ceramide, a lipid capable of inducing apoptosis (25), compared with littermate controls (Figure 7A). This was associated with increased expression of long chain base 1 (LCB1) and LCB2, the subunits of serine palmitoyl-CoA transferase. Expression of apoptosis-related genes — B cell leukemia/lymphoma 2 (Bcl2), Bcl-2-like protein (Bcl-XL), BCL2-associated X protein (Bax), and caspase-9 — also dramatically increased (Figure 7B). The CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), one of the regulators of ER stress–mediated cell death, was upregulated in the MHC-PPARγ1H mice. iNOS was unchanged. There was evidence of positive TUNEL staining of heart tissue in both MHC-PPARγ1L and MHC-PPARγ1H, but not control, hearts (Figure 7, C and D).
Lipoapoptosis in MHC-PPARγ1 hearts. (A) Cardiac ceramide was measured in heart tissue from 4-month-old MHC-PPARγH male mice and their littermate controls using diacylglycerol kinase assay and normalized for tissue weight (n = 3–6). (B) Ceramide synthesis genes (LCB1 and LCB2), ER stress–induced apoptosis-related gene (CHOP), and apoptosis genes (Bax and caspase-9) were upregulated in MHC-PPARγ1H male mice hearts. Antiapoptosis genes Bcl2 and Bcl-XL were also upregulated (n = 8–9). (C) Cardiac ventricular tissues from control (left panel) and 8-month-old MHC-PPARγ1L (middle panel) and 4-month-old MHC-PPARγ1H male mice (right panel) were stained for DNA fragmentation by a TUNEL protocol (original magnification, ×200). Apoptotic cells containing fragmented nuclear chromatin exhibited a dark brown nuclear staining. (D) The TUNEL-positive myocytes were counted and expressed as the number of TUNEL-positive myocytes per millimeter squared tissue area. Data are shown as mean (± SD) and normalized to values for littermate controls (set as 1.0). *P < 0.05, **P < 0.01, and ***P < 0.001 versus littermate controls.
PPARγ1 expression in humans and streptozotocin-induced diabetic mouse hearts. We then tested whether PPARγ expression could be induced in the hearts of diabetic mice. Hearts of diabetic mice had 2.1-fold higher expression of PPARγ than those of control mice (P = 0.003) (Figure 8A). We also assessed PPARγ expression in hearts from humans. Human hearts had 8.1- to 14.5-fold higher PPARγ mRNA levels than mouse hearts (Figure 8B). These data suggest that PPARγ is more functional in human hearts.
Upregulation of PPARγ1 expression in streptozotocin-induced diabetic mouse and humans hearts. (A) Four-month-old wild-type C57BL/6 male mice were treated with streptozotocin (STZ), and 3 weeks later hearts from control and diabetic mice (glucose >300 mg/dl) were harvested and PPARγ1 mRNA assayed by qRT-PCR. (B) Heart tissue was obtained from a normal individual (Normal Hu) and from hearts of patients undergoing heart transplantation (Hu Tx). PPARγ1 and 18S rRNA were measured in human and wild type mouse mRNAs using primers that were common for human and mouse genes. ***P < 0.001 versus mice group.