Metallothionein Prevents High-Fat Diet–Induced Cardiac Contractile Dysfunction : Role of Peroxisome Proliferator–Activated Receptor γ Coactivator 1α and Mitochondrial Biogenesis Free

The experimental procedure described here was approved by our institutional animal use and care committee. In brief, 4-month-old male FVB and cardiac-specific metallothionein overexpression transgenic mice weighing ∼20 g were randomly assigned to low-fat (10% of total calorie) or high-fat (45% of total calorie) diets (Research Diets, New Brunswick, NJ) for 5 months. The high-fat diet was calorically rich (4.83 vs. 3.91 kcal/g in low-fat diet) due to higher fat composition. However, the two diets possessed similar nutrient composition. Mice were housed individually in a climate-controlled environment with a 12-h light/dark cycle and free access to diets and water. Fur color was used as a marker for metallothionein (dark brown) or FVB (white) mouse identification. After 5 months of feeding, all mice were fasted for 12 h and then given an intraperitoneal injection of glucose (2 g/kg body wt). Blood samples were drawn from the tail immediately before the glucose challenge, as well as 15, 60, and 120 min thereafter. Serum glucose levels were determined using an Accu-Chek III glucose analyzer (15). Systolic and diastolic blood pressure were examined with a semi-automated, amplified tail-cuff device. Blood insulin levels were measured using a mouse insulin enzyme-linked immunosorbent assay kit.

Cardiac geometry and function were evaluated in anesthetized (Avertin 2.5%, 10 μl/g body wt i.p.) mice using a two-dimensional guided M-mode echocardiography (Sonos 5500) equipped with a 15–6 MHz linear transducer. Anterior and posterior wall thickness and diastolic and systolic left ventricular dimensions were recorded from M-mode images using the method adopted by the American Society of Echocardiography. Fractional shortening was calculated from end-diastolic diameter (EDD) and end-systolic diameter (ESD) using the following equation: (EDD − ESD)/EDD. Heart rates were averaged over 10 cardiac cycles (16).

After ketamine/xylazine sedation, hearts were removed and perfused with Krebs-Henseleit bicarbonate (KHB) buffer containing (in mmol/l): 118 NaCl, 4.7 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 25 NaHCO₃, 10 HEPES, and 11.1 glucose. Hearts were digested with collagenase D for 20 min. Left ventricles were removed and minced before being filtered. Myocyte yield was ∼75% and was not affected by high-fat diet or metallothionein. Only rod-shaped myocytes with clear edges were selected for mechanical and intracellular Ca²⁺ study (15).

Mechanical properties of cardiomyocytes were assessed using an IonOptix soft-edge system (IonOptix, Milton, MA). Myocytes were placed in a chamber mounted on the stage of an Olympus IX-70 microscope and superfused (∼2 ml/min at 25°C) with a Krebs-Henseleit bicarbonate buffer containing 1 mmol/l CaCl₂. Myocytes were field stimulated at 0.5 Hz unless otherwise stated. Cell shortening and relengthening were assessed including peak shortening, indicating peak contractility; time-to-PS (TPS), indicating contraction duration; time-to-90% relengthening (TR₉₀), indicating relaxation duration; and maximal velocities of shortening/relengthening (±dL/dt), indicating maximal pressure development and decline (15).

A cohort of myocytes was loaded with fura-2/AM (0.5 μmol/l) for 10 min, and fluorescence intensity was recorded with a dual-excitation fluorescence photomultiplier system (Ionoptix). Myocytes were placed onto an Olympus IX-70 inverted microscope and imaged through a Fluor ×40 oil objective. Cells were exposed to light emitted by a 75W lamp and passed through either a 360- or a 380-nm filter, while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm, and qualitative change in fura-2 fluorescence intensity (FFI) was inferred from the FFI ratio at the two wavelengths (360/380). Fluorescence decay time (single or bi-exponential decay) was calculated as an indicator of intracellular Ca²⁺ clearing (15).

Production of cellular ROS was evaluated by analyzing changes in fluorescence intensity resulting from oxidation of the intracellular fluoroprobe DCF [5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate]. In brief, cardiomyocytes were loaded with 10 μmol/l DCF at 37°C for 30 min. The myocytes were rinsed, and the fluorescence intensity was then measured using a fluorescent microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 530 nm. Untreated cells showed no fluorescence and were used to determine background fluorescence (17).

Left ventricles were fixed with 2.5% glutaraldehyde/1.2% acrolein in fixative buffer (0.1 mol/l cacodylate, 0.1 mol/l sucrose, pH 7.4) and 1% osmium tetroxide, followed by 1% uranyl acetate, and dehydrated through a graded series of ethanol concentrations before being embedded in LX112 resin (LADD Research Industries, Burlington, VT). Ultra-thin sections (∼50 nm) were cut on the ultramicrotome, stained with uranyl acetate followed by lead citrate, and viewed on a Hitachi H-7000 Transmission Electron Microscope equipped with a 4K × 4K cooled charge-coupled device digital camera (18). Quantitative analyses of mitochondrial size and density were carried out at a magnification of ×4,000. An average of six to seven visual fields was evaluated for each mouse heart.

The protein was prepared as described (15). A subset of cardiomyocytes from FVB and metallothionein mice was first incubated at 37°C with free fatty acid palmitic acid (1 mmol/l) (see online supplementary methods [available at http://dx.doi.org/10.2337/db06-1596] for palmitic acid preparation) for 24 h before PGC-1α expression was determined. Samples containing equal amount of proteins were separated on 10% SDS-polyacrylamide gels in a minigel apparatus (Mini-PROTEAN II; Bio-Rad) and transferred to nitrocellulose membranes. The membranes were blocked with 5% milk in Tris-buffered saline with Tween and were incubated overnight at 4°C with anti-Akt, anti-pAkt, anti-Foxo1a, anti-pFoxo1a (Thr24), anti–silent information regulator (Sirt), anti-Foxo3a, anti-pFoxo3a (Thr32), PGC-1α (all at 1:1,000), and anti–β-actin (1:5,000). After immunoblotting, the film was scanned and the intensity of immunoblot bands detected with a Bio-Rad calibrated densitometer.

Total RNA was isolated from left ventricles using the TRIzol reagent (Invitrogen), followed by DNase digestion to eliminate genomic DNA contamination. RNAs were quantified with spectrophotometer A260 readings. Synthesis of cDNA was performed at 37°C for 60 min using 1 μg total RNA in a 20-μl system by Superscript III (Invitrogen). Primers were designed using Beacon Designer 5.0 software. The primers used were as follows: mouse nuclear respiratory factor (NRF)1: sense 5′-CCATCACAGACCGTAGTACAGAC-3′, antisense 5′-TCCATCAGCCACAGCAGAG-3′; NRF2: sense 5′-ATAGTTACCATTGACCAGCCTGTG-3′, antisense 5′-GACCATTGTTTCCTGTTCTGTTCC-3′; and fTAM: sense 5′-AGTCCATAGGCACCGTATTG-3′, antisense 5′-TTAGCACGCTCCACATTCC-3′. The primers for housekeeping gene GAPDH (mouse) were: sense 5′-AATGGTGAAGGTCGGTGTGAAC-3′, antisense 5′-CGTGAGTGGAGTCATACTGGAAC-3′. The real-time PCR was performed by using an iCycler iQ real-time PCR detection system (Bio-Rad) with a SYBR Green qPCR SuperMixes kit (Invitrogen). The thermocycling program was 40 cycles of 95°C for 15 s and 55°C for 45 s with an initial cycle of 95°C for 10 min. The melting curve analysis was performed over the range 55–95°C by monitoring SYBR Green fluorescence with increasing temperature (0.5°C increment with a 10-s interval). PCR-specific products were determined as a clear single peak at the melting curves >80°C. Real-time PCR was duplicated for each cDNA sample. Each gene mRNA level was acquired from the value of the threshold cycle (Ct) of the real-time PCR as related to that of GAPDH using the comparative Ct method through the formula 2 ΔCt (ΔCt = GAPDH Ct − gene of interest Ct) (19).

The relative amount of heart mtDNA copy number was determined using RT-PCR as described (20). Total DNA was extracted using a Qiagen DNeasy tissue kit; 10 ng DNA was used, and mitochondrial nicotinamide adenine dinucleotide dehydrogenase-5 (ND-5) was the target gene. Primers for ND-5 were: forward 5′TGG ATG ATG GTA CGG ACG AA-3′, reverse 5′-TGC GGT TAT AGA GGA TTG CTT GT-3′. Qualitative RT-PCR was performed using a BioRad real-time thermocycler coupled with SYBR Green technology and the following cycling parameters: stage 1, 50°C for 2 min; stage 2, 95°C for 10 min; and stage 3, 40 cycles at 95°C for 15 s and at 55°C for 45 s. Each sample was analyzed in duplicate. Relative mitochondrial copy number to nuclear copy number was assessed by a comparative threshold cycle method, using β-actin as the internal control.

Data are presented as means ± SEM. Statistical comparison was performed by ANOVA, followed by Newman-Keuls post hoc test. Significance was set at P < 0.05.

High-fat feeding significantly increased body but not organ (liver and kidney) weight compared with low-fat–fed mice. Metallothionein did not affect body or organ weights regardless of the diet regimen. Plasma insulin levels were significantly elevated equally in high-fat diet–fed FVB and metallothionein mice. Blood pressure (both diastolic and systolic), heart rate, wall thickness, left ventricular EDD, and normalized left ventricular mass were comparable among all mouse groups. High-fat feeding significantly increased ESD and left ventricular mass (indication of cardiac hypertrophy) and reduced fractional shortening, which was abrogated by metallothionein transgene. Metallothionein itself did not affect myocardial geometry and fractional shortening (Table 1). Following acute intraperitoneal glucose challenge, serum glucose levels in low-fat–fed FVB or metallothionein mice started to decline after peaking at 15 min and nearly returned to baseline after 120 min. However, postchallenge glucose levels remained at much higher levels between 15 and 120 min in high-fat–fed FVB and metallothionein mice, indicating glucose intolerance. Basal fasting glucose levels were comparable among all groups, excluding the existence of full-blown diabetes. Furthermore, high-fat–fed FVB mouse cardiomyocytes displayed a significantly elevated ROS accumulation, the effect of which was ablated by the metallothionein transgene (Fig. 1).

Neither high-fat diet nor metallothionein affected resting myocyte length. High-fat feeding significantly reduced peak shortening and ± dL/dt, as well as prolonged TR₉₀, without affecting TPS in cardiomyocytes of FVB mice, somewhat reminiscent of our earlier finding (8). Importantly, metallothionein abolished high-fat diet feeding–induced mechanical abnormalities (Fig. 2). In addition, cardiomyocytes from high-fat–fed mice displayed a significantly elevated baseline intracellular Ca²⁺, depressed intracellular Ca²⁺ rise in response to electrical stimulus (ΔFFI), and reduced intracellular Ca²⁺ decay rate (either single or bi-exponential curve fit). Metallothionein negated high-fat diet–induced prolongation in intracellular Ca²⁺ decay and depression in ΔFFI with little effect on elevated baseline FFI. Metallothionein itself did not affect any of the intracellular Ca²⁺ properties tested (Fig. 3).

Mouse hearts beat at high frequencies (>400/min at 37°C). To investigate possible derangement of cardiac excitation-contraction coupling at higher frequencies, the stimulating frequency was increased stepwise from 0.1 to 5 Hz (300 beat/min). Cells were initially stimulated to contract at 0.5 Hz for 5 min to ensure steady state before commencing the frequency protocol. All recordings were normalized to the peak shortening obtained at 0.1 Hz of the same cell. Myocytes from the high-fat–fed group exhibited significantly exaggerated depression in peak shortening at 3.0 and 5.0 Hz without change at low frequencies. Metallothionein transgene itself exerted little effect at all frequencies tested. However, it abrogated high-fat diet–induced depression in peak shortening at 3.0 and 5.0 Hz (Fig. 3E).

Without high-fat diet treatment, no ultrastructural difference was noted in cardiac samples between FVB and metallothionein groups (Fig. 4A and B). High-fat feeding triggered extensive focal damage in hearts of FVB mice characterized by mitochondrial swelling, disorganization of cristae, and loss of sarcomere integrity (Fig. 4C). Consistent with the mechanical observation, metallothionein negated high-fat diet–induced cardiac structural damage (Fig. 4D). Myocardial tissues from high-fat diet–fed metallothionein mice were ultrastructurally indistinguishable from low-fat diet groups. Quantitative analysis revealed that high-fat diet significantly reduced the density but not the size of mitochondria in hearts of FVB mice, the effect of which was nullified by metallothionein (Fig. 4E and F).

To explore the possible mechanism involved in high-fat diet–and metallothionein-induced myocardial morphological and functional responses, the impact of high-fat diet and metallothionein on expression of the cardiac survival factor Akt, the mitochondrial biogenesis regulator PGC-1α, and the caloric-sensitive molecule Sirt was examined. Our results indicated that high-fat diet did not affect expression of Sirt, total and phosphorylated Akt, or pAkt-to-Akt ratio. Metallothionein did not elicit any significant effect on these proteins. Interestingly, high-fat diet significantly downregulated the nuclear receptor coactivator for mitochondrial biogenesis PGC-1α by ∼55%. Consistent with its effect on mitochondrial density, metallothionein ablated high-fat diet–induced reduction in PGC-1α (Fig. 5). Further study revealed that high-fat diet–and metallothionein-elicited effects on PGC-1α were mirrored by changes in the PGC-1α downstream factors including NRF1, NRF2, and mitochondrial transcription factor A (mtTFA). While high-fat diet significantly downregulated the levels of NRF1, NRF2, and mtTFA by 70, 40, and 39%, respectively, metallothionein effectively nullified high-fat diet–induced depression of these PGC-1α downstream nuclear factors essential for mitochondrial biogenesis (21) without any effect by itself. The mtDNA copy number, a key index for oxidative mitochondrial damage and mitochondrial biogenesis, was reduced following high-fat diet feeding, the effect of which was reversed by metallothionein (Fig. 6).

To examine the causality in metallothionein-elicited protection against high-fat diet–induced downregulation of PGC-1α, we performed an in vitro study to incubate cardiomyocytes with palmitic acid (1 mmol/l) for 24 h. Our immunoblots revealed that palmitic acid significantly downregulated PGC-1α expression in FVB but not in metallothionein mice (Fig. 5F), suggesting that free fatty acids may directly downregulate the mitochondrial biogenesis coactivator and play a role in high-fat diet–elicited effects on PGC-1α.

PGC-1α is known to interact with a number of signaling pathways including the Forkhead transcriptional factors, which may be coactivated by PGC-1α in the regulation of gluconeogenic and heme biosynthetic genes (21). Evaluation in the expression of the Forkhead transcriptional factors Foxo1a and Foxo3a revealed that high-fat diet feeding significantly enhanced Foxo3a phosphorylation without affecting total protein expression of Foxo1a and Foxo3a, as well as the phosphorylation of Foxo1a. Metallothionein failed to alter either the total or phosphorylated form of both Forkhead transcriptional factors under low- or high-fat regimen (Fig. 7).

The major findings of our study are that high-fat diet feeding elicits glucose intolerance, cardiac hypertrophy, enhanced ROS accumulation, myocardial contractile dysfunction, impaired intracellular Ca²⁺ handling, mitochondrial damage, and reduced mitochondrial density. The high-fat diet–induced myocardial geometric, functional, and mitochondrial aberrations were associated with depression of mtDNA copy number, the critical mitochondrial activator PGC-1α, as well as its downstream nuclear factors NRF1, NRF2, and mtTFA, indicating a likely role of PGC-1α and its downstream nuclear factors in the reduced mitochondrial biogenesis and myocardial dysfunction under high-fat diet feeding. Intriguingly, the high-fat diet–induced myocardial geometric, functional, and mitochondrial aberrations were significantly attenuated or nullified by metallothionein. Our in vitro study revealed that palmitic acid, a main free fatty acid component in high-fat diet, downregulates PGC-1α expression in cardiomyocytes, the effect of which was ablated by metallothionein. More importantly, high-fat diet–induced depression in mtDNA copy number, PGC-1α, and its downstream nuclear factors NRF1, NRF2, and mtTFA were all restored by metallothionein, consolidating a role of these mitochondrial regulators in high-fat diet–induced cardiac damage and antioxidant-offered cardioprotection. Since metallothionein did not have any “metabolic effect” on glucose metabolism, plasma insulin, or body weight gain, our data suggest that the antioxidant-elicited beneficial effects against high-fat diet–induced myocardial contractile dysfunction and intracellular Ca²⁺ derangement likely result from cardiac-specific effects, including improvement of mitochondrial biogenesis.

High-fat diet intake leads to dyslipidemia, hyperinsulinemia, insulin resistance, obesity, and type 2 diabetes (9,22), all of which trigger cardiac contractile dysfunction via sympathetic activation, renin-angiotensin system stimulation (1,3). Although pharmacological intervention and lifestyle modification including caloric restriction, weight loss, and physical exercise have shown promise in improving the compromised cardiac function in obesity and insulin resistance (3,23), no therapeutic regimen can radically alleviate the devastating heart problems in obese individuals. Our current observation of compromised myocardial and cardiomyocyte contractile function (reduced fraction shortening, enlarged ESD, depressed peak shortening and ± dL/dt, and prolonged TR₉₀) following high-fat diet intake is consistent with previous findings from both human and experimental obesity (3,7,8,24). In addition, these high-fat diet–induced mechanical defects are reminiscent to those reported in full-blown type 1 and type 2 diabetes (2,25,26). However, our 5-month high-fat diet feeding elicited hyperinsulinemia and glucose intolerance with little changes in blood pressure and fasting blood glucose, not favoring contribution of concomitant hypertension and diabetes. The impaired intracellular Ca²⁺ handling shown as elevated resting intracellular Ca²⁺ levels, reduced intracellular Ca²⁺ clearance rate, and reduced intracellular Ca²⁺ rise (ΔFFI) in high-fat diet–fed myocytes of FVB mice is in line with data from a rat model of high-fat diet–induced obesity (8) and is likely responsible for prolonged relaxation, reduced peak shortening, and fraction shortening in hearts of high-fat diet–fed mice. Our observation of exaggerated peak shortening decline at high stimulus frequencies in high-fat–fed mice further supported dampened intracellular Ca²⁺ cycling capacity. The fact that metallothionein reconciled high-fat diet–induced intracellular Ca²⁺ mishandling and intracellular Ca²⁺ cycling suggests an essential role of intracellular Ca²⁺ homeostasis in high-fat diet–induced myocardial dysfunction and antioxidant-offered protection, consistent with our earlier report in a sucrose diet–induced insulin resistance model (15).

Perhaps the most significant finding from our study is that metallothionein restored high-fat diet–induced mitochondrial damage and reduced mitochondrial density, mtDNA copy number, and downregulation of mitochondrial biogenesis factor PGC-1α and its downstream nuclear factors. Reduced mitochondrial biogenesis and downregulation of genes required for mitochondrial oxidative phosphorylation have been implicated in high-fat diet intake, insulin resistance, and type 2 diabetes (9,27). PGC-1α may induce mitochondrial genes by enhancing levels of NRF1 and NRF2, alternatively called GA-binding protein (GABP), and facilitate GABP binding to regulatory regions of mtTFA promoter (21). It is reasonable to speculate that high-fat diet–induced downregulation of PGC-1α and its downstream nuclear factors (NRF1, NRF2, and mtTFA) may underscore reduced mitochondrial biogenesis and mitochondrial density. This is consolidated by our observation that palmitic acid directly downregulates expression of PGC-1α in cardiomyocytes from FVB but not metallothionein mice. Levels of palmitic acid, the predominant saturated free fatty acids released from adipose tissue, are usually elevated in inflammatory conditions such as obesity, insulin resistance, and diabetes and play an essential role in cardiovascular complications including mitochondrial damage and cardiac dysfunction under these disease states (28). PGC-1α seems to be essential for normal heart function as PGC-1α knockout or deficiency results in diminished cardiac function and heart failure (21,29), suggesting that decreased PGC-1α expression is maladaptive in heart disease. The cellular mechanism responsible for downregulated PGC-1α following high-fat diet intake is not fully clear, although posttranslational modification such as oxidative stress may play a role (21). It was demonstrated that deacetylation of PGC-1α through Sirt increases PGC-1α activity on hepatic gluconeogenic gene transcription (21,30), although our data did not favor any role of Sirt in the regulation of PGC-1α. The observation that antioxidant metallothionein rescues downregulated PGC-1α and its downstream nuclear factors suggests a role of oxidative stress in the regulation of PGC-1α, consistent with our ROS data. Metallothionein has been shown to offer protection against diabetic cardiomyocyte dysfunction through its antioxidant action (26,31). Nonetheless, further study is warranted to define the mechanism responsible for high-fat–and antioxidant-induced regulation on PGC-1α expression.

In our present study, high-fat diet induced Foxo3a phosphorylation without changes in Akt cascade, Foxo1a phosphorylation, and total Foxo expression. Akt is an essential cardiac survival factor for maintenance of cardiac contractile function (32). Nonetheless, our data did not favor any major role of Akt and its downstream signal Foxo in metallothionein-offered cardioprotection against high-fat diet intake. Although our observation of enhanced Foxo3a phosphorylation appears to coincide with cardiac hypertrophy under high-fat intake due to inhibition of Forkhead-mediated apoptosis, the fact that metallothionein protects cardiac hypertrophy and myocardial dysfunction against high-fat intake without affecting elevated pFoxo3a levels suggests the contribution of alternate or compensatory mechanisms. In fact, the mitochondrial regulator PGC-1α has been shown recently to suppress Foxo3 transcriptional factor and participate in lysosomal proteolysis and protein degradation (14). However, our study did not identify the interaction between PGC-1α and Forkhead transcription factors.

Concerning experimental limitations, ideally, mice with various pharmacologically or genetically altered metallothionein levels should help to demonstrate whether any robust relationship among high-fat diet feeding, mitochondrial integrity, and myocardial function exists. However, transgenic mice with different metallothionein levels are not readily available, while the pharmacological induction of metallothionein using zinc is rather nonspecific or potentially toxic. Our in vitro incubation of free fatty acid palmitic acid may not truly reflect the metabolic changes elicited by chronic high-fat diet feeding. Due to technical difficulty, we were unable to assess murine cardiomyocyte function following 24 h of palmitic acid treatment, which would provide essential information for high-fat diet–induced myocardial defect. Last but not the least, it was recently reported that resveratrol improves mitochondrial function through decreased PGC-1α acetylation and consequently increased PGC-1α activity (33). However, our results did not favor any role of the protein deacetylase Sirt in high-fat diet–and metallothionein-elicited response, indicating that disparate mechanisms may be present for PGC-1α regulation.

In conclusion, our study offered evidence for the first time that myocardial contractile dysfunction, intracellular Ca²⁺ mishandling, ROS accumulation, mitochondrial damage, and loss of mitochondrial density and mtDNA content in high-fat diet–induced obesity are closely associated with downregulation of mitochondrial biogenesis coactivator PGC-1α and its downstream nuclear factors. In light of metallothionein-elicited protection against high-fat–or palmitic acid–induced cardiac dysfunction, as well as downregulation of the master regulator of mitochondrial biogenesis PGC-1α and its downstream nuclear factors, our data support the novel hypothesis that high-fat diet intake (possibly insulin resistance and type 2 diabetes) impairs mitochondrial biogenesis, which may explain the pathogenesis of aberrant myocardial function under obesity, insulin resistance, and full-blown diabetes. It is imperative that we understand the regulation of PGC-1α and mitochondrial biogenesis under oxidative stress and antioxidant therapy so that treatment strategy may be aimed at achieving sufficient induction of PGC-1α within a therapeutically beneficial window.

This work was supported in part by the American Heart Association Pacific Mountain Affiliate (no. 0355521Z) and the American Diabetes Association (7-0-RA-21) to J.R.

This work was presented in abstract form at the 45th annual meeting of the American Heart Association, Chicago, Illinois, 12–15 November 2006.

The authors thank Dr. D.P. Thomas and C.X. Fang for assistance.