A Murine Model of Isolated Cardiac Steatosis Leads to Cardiomyopathy

doi:10.1161/HYPERTENSIONAHA.110.160655

Hypertension. Author manuscript; available in PMC 2012 April 10.

Published in final edited form as:

Hypertension. 2011 February; 57(2): 216–222.

Published online 2011 January 10. doi: 10.1161/HYPERTENSIONAHA.110.160655

PMCID: PMC3322545

NIHMSID: NIHMS364163

A Murine Model of Isolated Cardiac Steatosis Leads to Cardiomyopathy

Denis J. Glenn, Feng Wang, Minobu Nishimoto, Michelle C. Cruz, Yoshikazu Uchida, Walter M. Holleran, Yan Zhang, Yerem Yeghiazarians, and David G. Gardner

Department of Medicine and Diabetes Center (D.J.G., F.W., M.N., M.C.C., D.G.G.), Dermatology Service and Research Unit, Veterans Affairs Medical Center; Department of Dermatology (Y.U., W.M.H.); and Department of Medicine, Division of Cardiology (Y.Z., Y.Y.), University of California at San Francisco, CA

Correspondence to Denis J. Glenn, Diabetes Center, 1119 HSW, University of California at San Francisco, 513 Parnassus Ave, San Francisco, CA 94143-0540. Email: dglenn/at/diabetes.ucsf.edu

The publisher's final edited version of this article is available free at Hypertension

See other articles in PMC that cite the published article.

Abstract

Lipid accumulation in the heart is associated with obesity and diabetes mellitus and may play an important role in the pathogenesis of heart failure seen in this patient population. Stored triglycerides are synthesized by the enzyme diacylglycerol acyl transferase (DGAT). We hypothesized that forced expression of DGAT1 in the cardiac myocyte would result in increased lipid accumulation and heart dysfunction. A cardiac myocyte–selective DGAT1 transgenic mouse was created and demonstrated increased lipid accumulation in the absence of hyperglycemia, plasma dyslipidemia or differences in body weight. Over time, expression of DGAT1 in the heart resulted in the development of a significant cardiomyopathy. Echocardiography revealed diastolic dysfunction with increased early mitral inflow velocity to late mitral inflow velocity ratio and decreased deceleration time, suggesting a restrictive pattern in the transgenic mice. Moderate systolic dysfunction was also seen at 52 weeks. Histological analysis showed increased cardiac fibrosis and increased expression of procollagen type 1A, matrix metalloproteinase 2, and tissue inhibitor of matrix metalloproteinase 2 in the transgenic mice. Mitochondrial biogenesis was reduced in the transgenic hearts, as was expression of cytochrome c oxidase 1 and cytochrome c. Expression of key transcription factors important in the regulation of mitochondrial biogenesis were reduced. These findings suggest that triglyceride accumulation, in the absence of systemic metabolic derangement, results in cardiac dysfunction and decreased mitochondrial biogenesis.

Keywords: diacylglycerol acyltransferase, lipotoxicity, cardiomyopathy, cardiac fibrosis, mitochondrial biogenesis

Obesity is associated with the development of cardiomyopathy and in some cases overt heart failure that seems to be independent of comorbid conditions such as diabetes mellitus, hypertension, coronary artery disease, and dyslipidemia. Remarkably, obesity-related cardiomyopathy has been estimated to cause up to 11% of all cases of heart failure in men and up to 14% in women.¹ A frequent finding in the hearts of obese and diabetic patients is the presence of lipid inclusions within myocardial cells, a condition referred to as cardiac steatosis.^2–4 Some have suggested that this steatosis is toxic to the cardiac myocyte⁵ while others have argued that it may serve a protective function to neutralize the effects of fatty acids and their metabolites⁶ through esterification to neutral lipids.

In rodents, high-fat diet has been shown to result in both increased deposition of lipid within myocytes and cardiac dysfunction.^7,8 Myocardial lipid accumulation has also been observed in genetic models of obesity and insulin resistance.^3,9–11 Studies carried out using transgenic mice or mice with targeted gene deletions that selectively promote fatty acid uptake (eg, overexpression of acyl CoA synthase [ACS],¹² fatty acid binding protein,¹³ or lipoprotein lipase¹⁴) or that prevent lipid turnover (eg, deletion of the adipose triglyceride lipase gene¹⁵) each demonstrate increased myocyte lipid deposition and cardiac dysfunction. Collectively, these findings support the notion that increased lipid or fatty acid accumulation within the myocyte leads to structural and mechanical changes in the heart that compromise pump function.

Excess lipid in the heart is stored in droplets as neutral triglyceride,¹⁶ which is synthesized from fatty-CoA and diacylglycerol (DAG) in a reaction that is catalyzed by diacylglycerol acyl transferase (DGAT).¹⁷ Two gene products, DGAT1 and DGAT2, have been identified,^18,19 and tissue-specific transgenic expression of either DGAT1 or DGAT2 results in enhanced lipid accumulation.^20–23 To explore the effect of intracellular triglyceride on cardiac structure and function, we generated a murine model of cardiac steatosis by overexpressing DGAT1 selectively in the cardiac myocyte. This mouse displays both myocyte steatosis and progressive deterioration of myocardial structure and function over time.

Methods

An expanded Methods section is available in the online Supplement at http://hyper.ahajournals.org.

Results

DGAT1 Expression in the Heart

Expression of DGAT1 in rat neonatal cardiomyocytes resulted in lipid accumulation and activation of a B-type natriuretic peptide (BNP)-luciferase reporter gene, implying that enhanced DGAT1-mediated triglyceride synthesis and storage is associated with activation of the hypertrophic gene program (Figure S1 in the online Supplement). To extend these findings to an in vivo model, we created a transgenic mouse that selectively expresses DGAT1 in the cardiac myocyte. A line which showed moderate expression of the transgene was selected for further analysis (Figure S2). Expression of DGAT1 was quantified in the transgenic hearts and compared to nontransgenic (NTg) littermates (mice aged 12 weeks) by Q-PCR and Western Blot (Figure 1A and 1B). The latter demonstrated an approximately 2.5 fold increase in cardiac DGAT1 expression versus NTg controls. Selectivity of transgene expression was confirmed by Western blot using an antibody to the FLAG epitope. (Figure S2).

Figure 1

A cardiac myocyte selective MHC-DGAT1 Tg mouse. A, Q-PCR analysis of DGAT1 mRNA expression in heart and liver normalized for GAPDH. N = 4 each group. B, Western blot analysis of DGAT1 and GAPDH expression. Representative blot and pooled data are shown, (more ...)

DGAT1-mediated lipid accumulation in mice 12 weeks of age was demonstrated by enhanced oil red O staining in mice subjected to an overnight fast (Figure 1C). Triglycerides were quantified and the MHC-DGAT1 Tg hearts demonstrated a 1.8-fold increase over NTg littermates (Figure 1D). Cardiac free fatty acid and DAG levels were quantified in a similar fashion; however, there were no significant differences in these levels (Figure 1E and 1F) in extracts from NTg versus DGAT1 Tg hearts.

The serum lipid profile, fasting blood glucose, and the response to glucose or insulin challenge were unaltered in the 12-week-old MHC-DGAT1 Tg mice (Figure S3). Levels of phospho-Akt, a marker of insulin sensitivity in heart, were comparable in NTg versus MHC-DGAT1 Tg mice at baseline and following an insulin challenge (Figure S3). Collectively, these results suggest that systemic lipid and glucose homeostasis and cardiac insulin sensitivity are unchanged by the enhanced expression of DGAT1 in the heart.

DGAT1 Overexpression Is Associated With the Development of a Cardiomyopathy and Activation of the Hypertrophic Gene Program

DGAT1 Tg mice at 52 weeks of age demonstrated cardiac wall thickening and chamber dilation (Figure 2A and the Table). A subset of DGAT1 Tg mice aged 52 to 60 weeks displayed a gross increase in heart size and marked wall thinning and chamber dilation in H&E stained cross sections compared to littermate controls (Figure 2A). These mice demonstrated overt signs of heart failure (ie, piloerection, tachypnea, lethargy and ascites). Gravimetric analysis demonstrated no significant increase in biventricular weight, normalized for total body weight or tibial length, in MHC-DGAT1 Tg versus NTg mice suggesting wall thickening is limited in scope or transient as the hearts progress to dilated cardiomyopathy (Figure S4). Individual myocyte cross sectional area was quantified in cardiac sections stained with Wheat Germ Agglutinin-Fluorescein Iso-Thiocyanate (FITC-WGA) (Figure 2B). Compared to littermate controls the MHC-DGAT1 Tg hearts showed increased myocyte cross sectional area at 12 and 52 weeks compatible with hypertrophy of the individual myocytes (Figure 2C). Expression of atrial natriuretic peptide (ANP), BNP, and α-skeletal actin (αSA),²⁴ was enhanced in the MHC-DGAT1 Tg mouse hearts at 12 weeks of age (Figure 2D). Similar results were found at 32 and 52 weeks of age (data not shown).

Figure 2

Cardiomyopathy in MHC-DGAT1 Tg hearts. A, Representative NTg and MHC-DGAT1 Tg hearts at 12, 32, 52 weeks of age (top panel, bar represents 1 cm) and H&E-stained transverse sections (bottom panel, original magnification ×12.5; bar represents (more ...)

Table

Echocardiographic Measurements at 12, 32, and 52 Weeks

Cardiac Function Is Impaired in the MHC-DGAT1 Tg Mouse

Echocardiography was used to examine function in the MHC-DGAT1 Tg versus NTg mice (the Table and Figure S5). In mice aged 52 weeks (prior to the onset of external signs of heart failure, as described above) end diastolic (EDV) and systolic volumes (ESV) increased in both groups at 52 weeks, albeit to a larger degree in the transgenic mice. This increase in volume and presumably the accompanying increase in wall stress likely contributed to the heart dysfunction seen in the transgenic animals. There was a significant decrease in left ventricular ejection fraction (LVEF) in the MHC-DGAT1 Tg mice compared to NTg littermates.

Diastolic function was reduced at 12 weeks and continued to show impairment at 32 and 52 weeks in the MHC-DGAT1 Tg mice compared to the NTg mice. At each time point, MHC-DGAT1 Tg mice demonstrated a significant increase in the ratio of early mitral inflow filling velocity to late filling velocity (E/A ratio) and decreased deceleration time (DT), suggesting a restrictive pattern of diastolic dysfunction.²⁵

MHC-DGAT1 Hearts Demonstrate Increased Cardiac Fibrosis

Masson trichrome stained sections demonstrated cardiac fibrosis in the MHC-DGAT1 Tg mouse compared to NTg controls at 12 and 52 weeks of age (Figure 3A). Quantification of collagen volume showed a significant increase in the 12- and 52-week-old transgenic mice (Figure 3B). Procollagen type IA²⁶ was increased in left ventricular tissue from 12-week old MHC-DGAT1 Tg mice (Figure 3C). The lower migrating band (band B) seen on the procollagen type IA immunoblot was preferentially increased and may represent a processed form of procollagen which accompanies increased collagen biosynthesis.²⁷

Figure 3

MHC-DGAT1 Tg hearts demonstrate increased cardiac fibrosis. A, Representative NTg and MHC-DGAT1 Tg left ventricle stained with Masson’s trichrome at 12 and 52 weeks of age (original magnification ×200). B, Collagen volume fraction was (more ...)

As shown in Figure 3D, expression of matrix metalloproteinase (MMP2) and tissue inhibitor of matrix metalloproteinase (TIMP2) increased significantly in the hearts of 32-week-old MHC-DGAT1 Tg versus NTg mice. These results support the hypothesis that these transgenic hearts are actively remodeling as a result of DGAT1 overexpression.²⁸ MMP9 expression was not different in MHC-DGAT1 Tg versus NTg hearts.

Mitochondrial Content Is Reduced in MHC-DGAT1 Tg Hearts

Mitochondrial biogenesis and function are thought to be negatively impacted by obesity and type 2 diabetes mellitus²⁹ and mitochondrial dysfunction has been linked to cardiac dysfunction in a number of experimental settings.³⁰ Electron microscopy did not reveal obvious mitochondrial structural abnormalities in the transgenic hearts at 12 weeks (Figure 4A) or 52 weeks (data not shown). However, analysis of mitochondrial volume³¹ revealed a 32% reduction in the 12-week-old MHC-DGAT1 Tg hearts compared to those of the NTg littermates (Figure 4B). Using a Q-PCR based method³² to estimate the relative number of mitochondria per cell we found a significant reduction in the 12-week-old MHC-DGAT1 Tg hearts (Figure 4C). In similar fashion, expression of mitochondrial cytochrome c oxidase and cytochrome c was reduced in the transgenic versus NTg hearts (Figure 4D).

Figure 4

Mitochondrial biogenesis is reduced in MHC-DGAT1 Tg hearts at 12 weeks of age. A, Representative electron micrographs of myocardium from NTg and MHC-DGAT1 Tg mice, original magnification ×5800; bar represents 0.5 mm. Lipid droplets (LD) and mitochondria (more ...)

Mitochondrial biogenesis and function are regulated by several key transcription factors, including nuclear respiration factor 1 and 2 (NRF1 and NRF2) and mitochondrial transcription factor A (Tfam).³³ We examined the levels of both NRF1 and Tfam by Q-PCR and found a significant decrease in each gene product in the 12-week-old MHC-DGAT1 Tg (versus NTg) hearts (Figure 4E). In addition, expression of both peroxisome proliferator-activated receptor (PPAR)α³⁴ and PPARδ,³¹ which have been shown to be important in supporting mitochondrial function and biogenesis in the heart, were reduced in the transgenic hearts. The transcriptional cofactor PPARγ coactivator 1 (PGC1α) has been shown to be important in cardiac function.^35,36 It interacts with members of the PPAR family, as well as NRF1 and NRF2, which, as noted above, serve to control mitochondrial biogenesis and function. Interestingly, we saw a reduction of PGC1α mRNA (Figure 4E) and protein (Figure 4F) levels in the MHC-DGAT1 Tg hearts compared to controls. Thus, overexpression of DGAT1 and subsequent lipid accumulation is associated with a reduction in mitochondrial biogenesis that appears to be linked to a reduction in expression of transcription factors that control this process.

Discussion

We have shown that transgenic expression of DGAT1 results in cardiac dysfunction in the absence of obesity, elevations in plasma lipid levels, or insulin resistance. These results support the hypothesis that cardiac steatosis has detrimental effects on myocyte function independent of those effects that accrue from excessive generalized adiposity or elevated plasma lipid levels.

Our results are seemingly in contrast with previous studies of DGAT1 and lipid accumulation. In studies of isolated fibroblasts from DGAT1-null mice, treatment with the fatty acid oleate resulted in increased cell death, suggesting that the inability to esterify fatty acids into neutral triglycerides efficiently potentiates the ability of the former to promote cellular dysfunction.³⁷ In a recent study, Liu et al³⁸ independently demonstrated that cardiac transgenic expression of DGAT1 results in triglyceride accumulation. In contrast to our findings, cardiac function in their transgenic model was unaffected. They go on to show that in a double transgenic model, in which both DGAT1 and ACS are coexpressed, DGAT1 expression appears to be cardioprotective. In this model, DGAT1 expression improves cardiac function and results in reduced diacylglycerol and ceramide content, as compared to the single ACS transgenic mouse.

These seemingly disparate results may be explained, in part, by the time course of the observed responses. The effect of DGAT1 overexpression on cardiac function in the Liu et al study was assessed in mice aged 3 to 4 months. The MHC-DGAT1 Tg mice in our study demonstrate modest but significant cardiac phenotypic changes at 12 weeks of age but required a year to develop severe cardiomyopathy. One might interpret these data, in aggregate, as suggesting that DGAT1-dependent triglyceride synthesis can be cardio-protective in the face of acute or subacute fatty acid overload. In this model, enhanced expression of DGAT1 would provide a mechanism to sequester fatty acids and blunt their toxic potential. Indeed, it has been suggested that such a mechanism occurs in the exercise-induced accumulation of triglyceride in skeletal muscle, a phenomenon termed the athlete’s paradox.^23,39 However, with time and even in situations where there is little or no threat of toxicity from plasma fatty acids, the DGAT1-dependent accrual of triglycerides within myocyte vesicles would provide a chronic source of endogenous triglyceride metabolites and fatty acids that could result in cellular toxicity.

The MHC-DGAT1 Tg hearts displayed considerable interstitial fibrosis, a hallmark of advanced cardiomyopathy and heart failure in animal models⁴⁰ as well as human patients.⁴¹ It has also been observed in the hearts of Zucker fatty rats⁴² and ob/ob mice.⁴³ The current study suggests that this interstitial fibrosis and increased remodeling activity can result solely from cardiac steatosis in the absence of elevations in plasma lipid levels. It also implies that the changes observed in the interstitial compartment as the cardiomyopathy develops are dependent on abnormalities originating in the myocyte itself since the genetic lesion is confined to this compartment. This could represent an indirect lipotoxic effect of fatty acids that are hydrolyzed from myocyte triglycerides and released into the interstitial compartment. Alternatively, it could reflect release of autocrine/paracrine factors by the myocytes in response to lipotoxic injury that, in turn, influence neighboring cardiac fibroblasts to increase cellular proliferation and synthesis of extracellular matrix.

Lipotoxicity has been associated with mitochondrial dysfunction in other systems,⁴⁴ and mitochondrial biogenesis is thought to be adversely affected in obesity and type 2 diabetes mellitus.²⁹ Skeletal muscle biopsies in type 2 diabetes mellitus patients show reduced oxidative function and decreased mitochondrial area, findings that correlate with reduced insulin sensitivity.^45,46 Both the number and function of mitochondria in the heart are linked inversely to changes in cardiac output that occur in response to physiological or pathological stress.⁴⁷ We found a significant reduction in measures of mitochondrial number, expression of mitochondrial encoded genes, and key transcription factors known to be important in mitochondrial biogenesis in the MHC-DGAT1 Tg hearts. We also noted reduced expression of the transcriptional coactivator PGC1α, a protein which has been shown to play an important role in mitochondrial biogenesis and fatty acid metabolism.⁴⁸ PGC1α is reduced in both heart and skeletal muscle in a rat model of congestive heart failure,⁴⁹ and deletion of the PGC1α gene in 2 independently generated mouse lines results in a cardiomyopathic phenotype.^36,50 Expression of PGC1α target genes are also reduced in myocardium of human patients with heart failure.⁵¹ Our findings raise the intriguing possibility that lipid accumulation and the lipotoxicity that it engenders might impair mitochondrial biogenesis and contribute to the development of cardiomyopathy, at least in part, through suppression of PGC1α expression.

Perspective

We have used cardiac selective expression of DGAT1, a rate-limiting terminal enzyme in triglyceride synthesis, to create a murine model of isolated cardiac steatosis. Over time, these mice demonstrate increased myocardial lipid deposition and progress to cardiomyopathy with compromised systolic and diastolic function. The protracted time course for development of cardiomyopathy in the MHC-DGAT1 Tg hearts is noteworthy in that it suggests that cardiac steatosis has insidious yet steadily progressive effects on myocyte function reminiscent of the dysfunction seen in obesity-related⁵² or diabetic cardiomyopathy,⁵³ both of which have been associated with myocyte steatosis.^2,54 This supports the notion that chronic accumulation of neutral lipid within myocardial cells in morbid obesity and diabetes mellitus is not a benign process and may well contribute to the cardiomyopathy and heart failure seen late in the course of those diseases.

Supplementary Material

data supplement

Fig. S1. Expression of DGAT1 in neonatal cardiac myocytes results in lipid accumulation and enhanced BNP reporter activity. (A) Cardiac myocytes were transfected with an either an empty expression vector (CMV) or a vector expressing FLAG-DGAT1 and treated with BSA or BSA: palmitate (4:1 molar ratio). Lipid accumulation was visualized with ORO staining where red identifies neutral lipid. Expression of FLAG-DGAT1 was confirmed by immunocytochemistry (brown stain). (B) The −1595 bp BNP luciferase reporter was cotransfected with the FLAG-DGAT1 expression vector. Cultures were treated with BSA, BSA: acid palmitate (saturated fatty acid) or oleate (unsaturated fatty acid) at a 4:1 molar ratio. After 48 hours, cells were collected and lysates were prepared for measurement of firefly or Renilla luciferase activity. Pooled data from 3 independent experiments are presented as mean ± SEMs.

Fig. S2. Construction of a cardiac myocyte selective MHC-DGAT1 Tg mouse. (A) Representation of the MHC-Flag-DGAT1 Tg construct (left panel). The presence of the transgene was confirmed by Southern Blot analysis in 4 independent founders, and Line D was used for subsequent studies (middle panel). A PCR based screen was used to identify the transgene in subsequent generations (right panel). (B) Western blot analysis of expression of the FLAG-DGAT1 and GAPDH from protein extracts derived from heart, skeletal muscle, lung, kidney and liver in the NTg and MHC-DGAT1 Tg tissue. (C) Expression of DGAT1 in NTg and MHC-DGAT1 Tg heart sections using anti-FLAG (green, top panel) or anti-DGAT1 (red, bottom panel). As a control, anti-mouse Alexa fluor 488 and anti-rabbit cy3 fluorescent secondary antibodies were incubated with the MHC-DGAT1 Tg heart sections in the absence of primary antibody. Original magnification 200×. *p<0.05, ***p<0.001. All data represent mice aged 12 weeks.

Fig. S3. Body weight, blood glucose and serum lipid profile in NTg and MHC-DGAT1 Tg mice. (A) Body weight of female (F) and male (M) NTg (N=9 males and N=6 females) and MHC-DGAT1 Tg (N=4 males and N=5 females) littermates. No statistical differences were noted. (B) Plasma triglycerides (TG), total cholesterol (chol.) high density lipoprotein (HDL) and free fatty acid (FFA) in NTg and MHC-DGAT1 Tg mice. N=4 for each group. No statistical differences were noted. Fasting blood glucose, glucose tolerance (C) and insulin tolerance (D). (NTg, N=6, MHC-DGAT1, N=5). No statistical difference between groups was noted. Western blot analysis of phospho-Akt, total Akt and GAPDH from heart extracts derived from NTg and MHC-DGAT1 TG mice in control mice (E) and following insulin treatment (F). Phospho-Akt was quantified and normalized against total Akt. No significant differences were noted. N=4 each group. All data represent NTg and MHC-DGAT1 Tg mice aged 12 weeks.

Fig. S4. Assessment of blood pressure and cardiac hypertrophy. (A) Blood pressure, N=6 each group, mice aged 12 weeks. Systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP). No statistical differences between groups were noted. Biventricular weight was measured and normalized to (B) body weight (HW/BW, mg/g) and (C) tibial length (HW/TL, mg/mm) in mice aged 12 weeks (N=4 each group), 32 weeks (NTg, N= 5 and MHC-DGAT1 Tg N= 7) and 52 weeks (NTg, N =14 and MHC-DGAT1 Tg, N=6). No significant differences were noted.

Fig. S5. Evaluation of cardiac function in 52 week old NTg and MHC-DGAT1 Tg mice by M-mode and Doppler echocardiography. M-mode echocardiographic images (A and B) and transmitral blood flow (C and D) were obtained in NTg (A, C) and MHC-DGAT1 Tg (B, D) mice aged 52 weeks. Mitral inflow early filling velocity (E), mitral inflow late filling (atrial contraction) velocity (A), and deceleration time (DT). Representative images are shown.

Fig. S6. Apoptosis, ceramide and lipid peroxidation. (A) Representative TUNEL stained fixed ventricular sections. Nuclei were counterstained with hematoxylin. Arrows indicate positive stained nuclei. (B) The number of TUNEL positive nuclei was quantified using ImageJ software. *p < 0.05. (C) Quantification of extracted cardiac ceramide (N=5 each group). No significant difference was noted. (D) Lipid peroxidation as assessed by total malondialdehyde (MDA) + 4-hydroxyalkenals (4-HAE) concentration in NTg and MHC-DGAT1 Tg heart tissue extracts. N= 5 each group. No significant difference was noted. All data represent NTg and MHC-DGAT1 Tg mice aged 12 weeks.

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Acknowledgments

We thank Dr Philip Ursell for assistance with interpretation of the electron microscopic images.

Sources of Funding

Supported by ADA grant 1-05-RA-43 (DGG), NIH HL 45637 (DGG), NIH HL 086158 (DJG), F32 HL074643 (FW) and AHA Grants in Aid 0455050Y (DGG), 09GRNT2251091 (DGG), and 09BGIA2260022 (DJG).

Footnotes

Disclosures

None.

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