Aging impairs myocardial fatty acid and ketone oxidation and modifies cardiac functional and metabolic responses to insulin in mice

Abstract

Aging presumably initiates shifts in substrate oxidation mediated in part by changes in insulin sensitivity. Similar shifts occur with cardiac hypertrophy and may contribute to contractile dysfunction. We tested the hypothesis that aging modifies substrate utilization and alters insulin sensitivity in mouse heart when provided multiple substrates. In vivo cardiac function was measured with microtipped pressure transducers in the left ventricle from control (4–6 mo) and aged (22–24 mo) mice. Cardiac function was also measured in isolated working hearts along with substrate and anaplerotic fractional contributions to the citric acid cycle (CAC) by using perfusate containing ¹³C-labeled free fatty acids (FFA), acetoacetate, lactate, and unlabeled glucose. Stroke volume and cardiac output were diminished in aged mice in vivo, but pressure development was preserved. Systolic and diastolic functions were maintained in aged isolated hearts. Insulin prompted an increase in systolic function in aged hearts, resulting in an increase in cardiac efficiency. FFA and ketone flux were present but were markedly impaired in aged hearts. These changes in myocardial substrate utilization corresponded to alterations in circulating lipids, thyroid hormone, and reductions in protein expression for peroxisome proliferator-activated receptor (PPAR)α and pyruvate dehydrogenase kinase (PDK)4. Insulin further suppressed FFA oxidation in the aged. Insulin stimulation of anaplerosis in control hearts was absent in the aged. The aged heart shows metabolic plasticity by accessing multiple substrates to maintain function. However, fatty acid oxidation capacity is limited. Impaired insulin-stimulated anaplerosis may contribute to elevated cardiac efficiency, but may also limit response to acute stress through depletion of CAC intermediates.

shifts in cardiac substrate metabolism or preference accompany several physiological and pathological conditions, such as fasting, exercise, diabetes mellitus, myocardial ischemia, and heart failure (5, 19). Fatty acid oxidation rates generally decrease in relationship to glucose oxidation and carbohydrate utilization during the development of myocardial hypertrophy (31). Aging presumably initiates shifts in substrate oxidation, which resemble those noted in other models of cardiac hypertrophy and contribute to impaired contractile function. Several experimental and clinical studies have reported a decline in fatty acid oxidation in the aged heart, with a proportional increase in glucose oxidation (2, 15). McMillin et al. (22) showed that the senescent intact rat heart supplied with oleate lacked the ability to suppress glucose oxidation. In contrast, Sample et al. (27) showed that myocardial palmitate oxidation was significantly increased and lactate oxidation depressed in 24-mo-old, senescent Wistar rats compared with 6-mo-old rats. Those authors suggested that the discrepant results were related to omission of lactate in perfusate used in prior experiments. Koonen et al. (17) recently showed in isolated working mouse heart aged 50–52 wk an increase in fractional contribution of palmitate to the citric acid cycle (CAC) compared with younger mice, although with an overall decrease in palmitate oxidation under similar substrate supply limitations (only glucose and palmitate as substrates).

Accordingly, substrate oxidation and fractional contributions (Fc) of acetyl-CoA in the aging mouse heart are ill-defined and have not been evaluated previously under robust substrate supply conditions present in vivo. In particular, lactate and ketones provide major sources for oxidative substrate in vivo, both of which suppress oxidation of glucose, a relatively minor contributor to myocardial oxidation in vivo. The mouse heart is frequently used as a model to study cardiomyopathy related to aging. The first objective of this study was to clearly define aging-induced shifts in myocardial substrate utilization, particularly relative to contributions to the CAC by free fatty acids (FFA) and ketones.

Alterations in myocardial substrate oxidation in some aging models have been attributed to increased insulin resistance (4). In canines, aging promotes an increase in insulin-mediated myocardial uptake and storage of fat. Although insulin normally promotes storage of fatty acids as intracellular triglycerides in muscle (8), the findings in canines suggest the existence of aging-induced alterations in cellular lipid distribution. This occurs in the setting of elevated plasma nonesterified fatty acids (NEFA) and triglycerides. Therefore, it is unknown if the myocardium is responding to systemic abnormalities in lipid metabolism or if these abnormalities are intrinsic to the heart. Furthermore, age-related changes in insulin sensitivity have not been evaluated in the mouse heart, a frequent model used to evaluate metabolism and study effects of aging. Accordingly, the second objective was to test the hypothesis that aging alters insulin sensitivity in the aged mouse heart when provided a physiological supply of substrates.

MATERIALS AND METHODS

Animals.

This study used mice in two age groups with mixed genetic background (C57BL/6–129Svj) Control and aged groups consisted of mature 4- to 6- and 18- to 22-mo-old mice, respectively. This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Pub. No. 85-23, revised 1996). All animal procedures were reviewed and approved by the Animal Care Committee at Seattle Children's Hospital Research Institute.

In vivo hemodynamics.

A subset of mice from each group (control, n = 5; aged, n = 4) underwent anesthesia induction under a nose hood with isoflurane (4%), which was decreased to 0.8% for maintenance and under conditions of steady state. Left ventricular pressure and volume were measured with a PV-Catheter (SPR-839, Millar Pressure-Volume Systems) inserted through the carotid artery. All parameters were measured constantly over a 20-min period and averaged.

Isolated working heart preparation.

Mice were anesthetized with pentobarbital sodium (75 mg/kg ip) and heparinized (700 U/kg ip). The heart was rapidly excised and submerged in ice-cold physiological salt solution (PSS), pH 7.4, containing (in mmol/l) 118.0 NaCl, 25.0 NaHCO₃, 4.7 KCl, 1.23 MgSO₄, 1.2 NaH₂PO₄, 5.5 d-glucose, and 1.2 CaCl₂.

The aorta of a spontaneously beating heart was cannulated first in a standard Langendorff mode and perfused with PSS. After the heart function had stabilized (15 min), the mode was changed to working heart perfusion with the inflow tube inserted into the left atrium. The perfusate was switched to PSS containing the following ¹³C-labeled substrates in addition to unlabeled glucose (5.5 mmol/l): 1,3-[¹³C]acetoacetic acid (0.17 mmol/l), l-lactic-3-[¹³C]acid (1.2 mmol/l), and U-[¹³C]-long-chain mixed FFA (0.35 mmol/l) bound to 0.75% (wt/vol) delipidated bovine serum albumin reconstituted with deionized water. The FFA mixture contained primarily saturated and unsaturated fatty acids ranging from 14 to 22 carbons in length, with palmitic and linoleic as the most prominent. The FFA-to-albumin ratio was slightly higher than used by other investigators (20). Details regarding this preparation and labeling strategy have been published previously (13).

Perfusates were equilibrated with 95% O₂-5% CO₂ at 37°C and passed twice through filters with 3.0-μm pore size. Perfusion pressure was maintained at 70 mmHg with Langendorff mode. With working mode preload was 10 mmHg and afterload was 50 mmHg. The entire perfusion system was jacketed and maintained at 37°C.

An SPR-PV-Catheter (SPR-869 or -839, Millar Pressure-Volume Systems) was inserted into the left ventricle through the apex for continuous measurement of left ventricular pressure (LVP). Recorded parameters from the left ventricle in addition to LVP (mmHg) were heart rate (HR, beats/min), positive and negative change in pressure over time (±dP/dt, mmHg/s), and left ventricular volume (μl). Further calculations provided stroke volume, cardiac output, work, power, cardiac efficiency, (power/oxygen consumption), pressure-rate product (PRP), V_max, and P-V loop, etc., although only some of these are presented in the results.

A cannula in the pulmonary artery enabled collection of coronary flow. Aortic and coronary flow rates were measured with a flowmeter (T403; Transonic Systems, Ithaca, NY). The caudal vena cava, cranial vena cava, and azygous vein were ligated.

To characterize cardiac function, left ventricular developed pressure (LVDP) was defined as peak systolic pressure minus end-diastolic pressure. Myocardial oxygen consumption (MV̇o₂) was calculated as MV̇o₂ = CF × [(Pa_O2 − Pv_O2) × (c/760)] × dw, where CF is coronary flow (ml·min⁻¹·g wet tissue⁻¹), (Pa_O2 − Pv_O2) is the difference in the partial pressure of oxygen (Po₂, mmHg) between perfusate and coronary effluent, c is the Bunsen solubility coefficient of O₂ in perfusate at 37°C (22.7 μl O₂·atm⁻¹·ml⁻¹), and dw is a previously determined conversion factor from heart wet weight to dry weight (1/0.18). Po₂, Pco₂, and pH were determined with a ABL800 blood gas analyzer (Radiometer, Copenhagen, Denmark).

Experimental protocol.

Hearts were divided into four groups: 1) control group without insulin (Con, n = 8); 2) control group with insulin, 80 μU/ml in perfusate (Con-Ins, n = 7); 3) aged group without insulin (Age, n = 8); and 4) aged group with insulin (Age-Ins, n = 7). After a 15-min equilibration period in Langendorff mode, the working heart mode was established. The baseline recording was taken 15 min into working heart mode. Functional parameters were recorded continuously with the Millar Pressure-Volume Systems, and outflow samples for blood gas analyses were taken every 10 min. After 15 min of baseline recording, Con-Ins and Age-Ins hearts were infused with 80 μU/ml of insulin. After 30 min of ¹³C-labeled substrate perfusion, nonventricular tissue was removed and hearts were instantly freeze clamped with chilled copper tongs.

Extraction.

Briefly, freeze-clamped hearts were pulverized under liquid nitrogen, extracted with 0.6 M perchloric acid, and neutralized with cold KOH to pH 7.4. The final supernatant was lyophilized overnight at −50°C for NMR analysis.

¹³C NMR and isotopomer analyses.

Substrate metabolism was established by using ¹³C-labeled substrates in combination with NMR spectroscopy. Glutamate isotopomer analyses provide fractional contribution of acetyl-CoA to the CAC from up to three differentially labeled substrates and the unlabeled and anaplerotic components.

Lyophilized heart extracts were dissolved in 99.8% D₂O for decoupled ¹³C NMR spectral acquisition. Free-induction decays (FIDs) were acquired on a Varian Direct Drive (VNMRS) 600-MHz spectrometer (Varian, Palo Alto, CA) equipped with a Dell Precision 390 Linux workstation running VNMRJ 2.2C. The spectrometer system was outfitted with a Varian triple resonance salt-tolerant cold probe with a cold carbon preamplifier. A Varian standard one-dimensional carbon direct observe sequence with proton decoupling was used to collect data on each sample.

FIDs were baseline corrected, zero-filled, and Fourier transformed. All of the labeled carbon resonances (C₁–C₅) of glutamate were integrated with the Lorentzian peak fitting subroutine in the acquisition program (NUTS, Acorn NMR, Livermore, CA). The individual integral values were used as starting parameters for the CAC analysis-fitting algorithm tcaCALC (21). This algorithm provided the Fc for each substrate in the acetyl-CoA pool entering CAC. The absolute flux for the CAC and the oxidative flux for individual substrates were calculated as previously described (13, 18).

Plasma substrates.

Plasma nonesterified fats (NEFA), glucose, triglycerides, β-hydroxybutyrate, cholesterol, and total triiodothyronine (T₃) were obtained from arterial samples from fully anesthetized mice with standard assays. These assays were performed by the National Mouse Metabolic Phenotyping Core at the University of Washington and Vanderbilt University.

Immunoblotting.

Fifty micrograms of total protein extract from mouse heart tissue was electrophoresed along with two lanes of molecular weight size markers (Novex Sharp Pre-stained, Invitrogen) in 4.5% stacking and 10% resolving SDS-polyacrylamide gels. The gels were then electroblotted onto PVDF-Plus membranes. The Western blot was blocked for 1 h at room temperature with 5% nonfat milk in Tris-buffered saline (TBS) plus Tween 20 (TBST; 10 mM Tris·HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween 20), followed by an incubation with primary antibody diluted in the above blocking solution. After four 5-min washes with TBST, the membrane was incubated at room temperature for 1 h with the appropriate IgG secondary antibody conjugated to horseradish peroxidase (HRP). The membranes were rinsed four times for 5 min with TBST and visualized with enhanced chemiluminescence upon exposure to Kodak BioMax Light ML-1 film. Membranes were stripped by washing for 30 min with 100 mM 2-mercaptoethanol, 2% (wt/vol) SDS, 62.5 mM Tris·HCl, pH 6.7, at 70°C, followed by three 10-min washes with TBS. After stripping, the membrane was again blocked for 1 h as above and incubated with an actin rabbit polyclonal antibody (sc-1616-R) diluted 1:500 in blocking solution. The membrane was washed (as above), a goat anti-rabbit secondary antibody-HRP (sc-2313) was applied, and the remaining procedure as described above was followed. The actin was used as an internal reference to determine protein lane loadings.

The primary antibodies used in this study were actin (sc-1616-R), PCB (sc-46228), AKT1 (sc-1618), carnitine palmitoyltransferase I (CPT-I)-M (sc-20670), CPT-I (sc-31128), glucose transporter-4 (Glut-4) (sc-1606), peroxisome proliferator-activated receptor (PPAR)α (sc-9000) obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The malonyl-CoA decarboxylase (MCD) antibody (15265-1-AP) was purchased from Proteintech Group (Chicago, IL). Thyroid hormone receptor antibodies TRα1 (PA1-211A), TRα2 (PA1-216), and TRβ (PA1-213A) were purchased from Affinity BioReagents (Golden, CO). The pyruvate dehydrogenase kinase (PDK)2 and PDK4 antibodies were obtained as personal gifts from Dr. Robert Harris (Indiana University School of Medicine, Indianapolis, IN).

RESULTS

Cardiac function.

Consistent with studies performed in other mouse strains, aged mice showed elevated heart weight-to-body weight ratios (H/W) [aged (n = 14), H/W 1.04 ± 0.048; control (n = 16), H/W 0.827 ± 0.049; P = 0.017]. A small subset of mice underwent hemodynamic assessment in vivo in order to define appropriate loading conditions for the isolated working heart experiments. Table 1 illustrates in vivo hemodynamic parameters. HR and dP/dt in both groups were lower than some literature values obtained under different anesthetic and physiological conditions. In contrast, stroke volume and cardiac output in these studies were somewhat higher than those from prior reports (29, 30). Under similar conditions for control and aged, there were no significant differences in left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), or LVDP (LVSP − LVEDP). Maximum +dP/dt (+dP/dt_max) and −dP/dt (−dP/dt_max) were also similar between the two groups. These data supported the use of similar volume and pressure loading conditions for the isolated working heart experiments, with the recognition that the power of the study was inadequate to identify significant differences in these parameters in vivo. However, despite the small sample size and consistent with prior studies employing echocardiographic measures of cardiac function, aged hearts did show significant decreases in stroke volume and cardiac output (20).

Table 1.

Hemodynamic parameters in vivo

	Control (n = 5)	Aged (n = 4)
DP, mmHg	89.6 ± 3.1	86.8 ± 3.1
LVSP, mmHg	100.6 ± 3.7	95.9 ± 2.0
LVEDP, mmHg	11.0 ± 1.6	9.1 ± 2.5
Stroke volume, μl	35.3 ± 4.4	21.1 ± 3.8*
+dP/dtmax, mmHg/s	3,653 ± 128	3,112 ± 323
−dP/dtmax, mmHg/s	3,357 ± 101	3,045 ± 360
HR, beats/min	363 ± 6	403 ± 20
PRP (×1,000)	31.8 ± 1.4	34.8 ± 1.8
CO, ml/min	12.8 ± 0.3	8.5 ± 0.8*

Values are means ± SE. Hemodynamic parameters were obtained with a microtipped pressure-volume transducer in the left ventricle. CO, cardiac output; LVSP, left ventricular systolic pressure; LVEDP, left ventricular end-diastolic pressure, DP, developed pressure (LVSP − LVEDP); HR, heart rate; PRP, mean left ventricular pressure-rate product (mmHg × beats per min); +dP/dt_max, maximum positive change in pressure over time.

^*P < 0.05 vs. control.

The working hearts from aged mice exposed to similar preload and afterload conditions as younger counterparts did not display diminished systolic or diastolic function (Figs. 1 and 2) when not exposed to insulin. Insulin produced effects on contractile function, which differed between the age groups. +dP/dt_max, −dP/dt_max, and MV̇o₂ were decreased by insulin in control hearts, reflecting the drop in CF (Fig. 2). In contrast, the aged hearts responded dramatically to insulin with a drop in HR but substantial improvement in multiple other parameters including +dP/dt_max and developed pressure. In the aged hearts insulin significantly increased cardiac output and power without increasing MV̇o₂, thus dramatically improving contractile energy efficiency.

Fig. 1.

Functional parameters for control and aged hearts with or without insulin. Con, control (n = 8); Con-Ins, control with insulin (n = 7); Age, aged (n = 8); Age-Ins, aged with insulin (n = 7); dP/dt_max, maximum change in pressure over time.

Fig. 2.

Myocardial oxygen consumption (MV̇o₂) and functional parameters for control and aged hearts with or without insulin. Efficiency is defined as cardiac power/oxygen consumption. Group abbreviations are as in Fig. 1.

Fractional contribution.

Figure 3 shows Fc of acetyl-CoA to the CAC for each studied substrate. Aging reduced Fc for FFA and increased Fc for unlabeled substrate (predominantly glucose). Insulin reduced FFA Fc in both control and aged hearts by a similar proportion. In response to insulin, the aged mice showed a marked increase in Fc for unlabeled substrate and a small decrease in Fc for lactate, which did not occur in control mice. Insulin promoted a trend toward an increase in anaplerotic contribution in control mice (P = 0.058). The anaplerotic contribution did not differ between control and aged mice without insulin. However, under insulin perfusion anaplerosis was significantly lower in the aged mouse hearts.

Fig. 3.

Fractional contribution to the citric acid cycle (CAC) for free fatty acids (FFA), acetoacetate as ketone (ACAC), lactate (LAC), and unlabeled substrates (Unlab). YS represents the relative anaplerotic contribution to the CAC. Group abbreviations and numbers of subjects are as in Fig. 1.

Substrate flux.

Figure 4 shows calculated flux rates for each substrate. The modest decrease in MV̇o₂ corresponds to the reduction in total CAC flux noted in aged hearts. In particular, aging suppressed fatty acid and acetoacetate flux. Insulin significantly inhibited fatty acid flux in control and aged hearts. As MV̇o₂ differed only slightly among groups, variations in flux reflect primarily alterations in Fc to the CAC.

Fig. 4.

Flux rates for total CAC and individual substrates. Group abbreviations and numbers of subjects are as in Fig. 1, and substrate abbreviations are as in Fig. 3.

Plasma substrates.

Values for plasma substrates are shown in Table 2. Plasma FFA and cholesterol were lower in aged mice. There were no differences in β-hydroxybutyrate or triglycerides between groups. However, T₃ levels were significantly lower in the aged mice.

Table 2.

Plasma metabolite and T₃ concentrations

	Control	Aged
Glucose, mg/dl	197 ± 7	183 ± 6
TG, mg/dl	65 ± 8	100 ± 9
Cholesterol, mg/dl	180 ± 2	153 ± 3*
FFA, meq/l	3.02 ± 0.3	2.12 ± 0.2*
β-Hydroxybutyrate, mM/l	0.13 ± 0.02	0.16 ± 0.03
Total T3, ng/ml	0.87 ± 0.03	0.64 ± 0.02†

Values are means ± SE. Metabolites were obtained after 12-h fast. TG, triglycerides; FFA, free fatty acids; T₃, triiodothyronine.

^*P < 0.05,

^†P = 0.00006.

Protein expression.

Several key proteins regulating fatty acid oxidation were analyzed for content in these hearts (Fig. 5). Malonyl-CoA is a pivotal regulator of fatty acid oxidation and synthesis. MCD controls conversion of this metabolite to acetyl-CoA and thereby promotes fatty acid oxidation. MCD expression showed no significant change with aging. Similarly, expression for muscle-specific (m)CPT-I and total expression for CPT-I (including liver-specific CPT-I) did not change with aging in these murine hearts. Glut-4 and PDK2 were also similar between groups. Importantly, PDK4 expression was substantially reduced by aging, approximating near 20% of control levels. We also evaluated expression for several transcription factors, which regulate cardiac metabolism. Akt showed no difference between groups. However, PPARα expression was substantially suppressed in aged hearts. Content for thyroid receptor isoforms α1 and β, as well as the dominant-negative TRα2, was not significantly altered by aging.

Fig. 5.

Expression for proteins involved in regulation of fatty acid metabolism. All values are normalized to actin (n = 5 for each group). PPAR, peroxisome proliferator-activated receptor; PDK, pyruvate dehydrogenase kinase; TRα1, TRβ1, and TRα2, thyroid hormone receptor α1, β1, and α2; CPT-I, total carnitine palmitoyltransferase I (including liver-specific CPT-I); mCPT-I, muscle-specific CPT-I; PCB, pyruvate carboxylase; MCD, malonyl-CoA decarboxylase; Glut-4, glucose transporter-4. The Akt and actin membrane was stripped and reblotted for PDK4; hence the same actin lane is observed.

DISCUSSION

Our novel findings challenge accepted concepts regarding metabolic flexibility of the aging heart. The data demonstrate substantial and fairly specific impairments in myocardial fatty acid and ketone oxidation in the aging mouse heart. Previous studies attributed reductions in myocardial fatty acid flux to an overall decrease in oxygen consumption and CAC flux (17), thereby suggesting mitochondrial impairment. Cardiac function in those studies was diminished under conditions of limited substrate, supplied only as palmitate and glucose (17). Our data show that the aged murine heart exhibits metabolic plasticity and maintains cardiac function by accessing and oxidizing alternative substrates. Our experimental protocol, which includes provision of a robust substrate supply with mixed fatty acids, ketones, lactate, and glucose, unmasks this metabolic plasticity. Therefore, the heart maintains contractile function and compensates for marked impairment in fatty acid and ketone oxidation, which accompany cardiac hypertrophy with aging. The aging-induced impairment in ketone body utilization has been unrecognized. Such deficiency would be expected to occur along the chain of ketone metabolism either at succinyl-CoA-acetoacetate transferase or thiolase.

The reductions in myocardial fatty acid flux and oxidation in aged mice in our study occur in conjunction and are consistent with marked depression of PPARα protein expression. PPARα acts as a transcriptional regulator of multiple target genes, which control fatty acid and glucose oxidation. Therefore, modification in PPARα represents a potential mechanism to explain change in fatty acid flux during aging. Prior studies have also shown lower cardiac PPARα expression and activity in aged rats (14) or murine models of accelerated aging (26). The mechanisms promoting a decrease in PPARα in the aged mouse heart are likely multifactorial. Changes in the circulating lipid profile such as occurred in our aged mouse group can modify PPARα expression.

Modulation in PPARα activity in models of aging occurs with variable responses in target genes and proteins. We found no age-induced changes in multiple proteins typically associated with modifications in fatty acid flux in heart. For instance, mCPT-I, considered a primary direct regulator of fatty acid oxidation, did not decrease with age. Although mCPT-I-suppressed expression has been noted in hearts from aged Fischer rats (14), downregulation of mCPT-I mRNA did not occur in the senescence accelerated mouse also exhibiting marked reductions in PPARα (26). Lack of sensitivity of cardiac mCPT-I to PPARα deficiency in these murine models suggests that PPARα influences fatty acid metabolism through modulation of other genes. We found a severalfold decrease in PDK4 protein during aging. Depression of PDK4 protein levels in these aged mice is comparable to that observed in PPARα-null mice (10). PDK4 inhibits pyruvate dehydrogenase and glucose oxidation, thereby reciprocally promoting fatty acid oxidation according to the Randle hypothesis.

Thyroid hormone highly regulates PDK4 expression and interacts with PPARα in promotion of PDK4 mRNA (3, 10). These interactions in heart depend on thyroid state. In the euthyroid and hyperthyroid states, thyroid hormone regulation predominates and PPARα antagonism produces minimal PDK mRNA or protein response (6, 10). Although hypothyroidism does not depress PDK4 in the rat heart (12), effects of hypothyroidism on PDK4 expression in mouse heart have not been specifically studied. Transgenic expression of the dominant-negative thyroid hormone receptor Δ337T in heart does suppress PDK4 mRNA response to PPARα antagonism by WY14643 (6). The aged mice show modest but significant depression in circulating T₃ levels, although no alterations in expression for thyroid hormone receptors. Conceivably, relative hypothyroid state in these aged hearts exacerbates the impact of PPARα deficiency on PDK4 expression.

Impairment in fatty acid oxidation in the aged heart can result in myocardial lipid accumulation. Excess accumulation in the intramyocardial lipid pool is linked to maladaptive hypertrophy (9, 17, 23). However, no single study has measured both fatty acid transport and oxidation in the same experimental model of aging. Koonen et al. (17) showed markedly elevated myocardial triacylglycerol and long-chain acyl-CoA levels in aged hearts, which displayed hypertrophy and depressed palmitate oxidation. Moderation of hypertrophy and functional abnormalities occurred in aged mice with knockout of fat/CD36, the major transmembrane fat transporter, although improvement was not linked to changes in myocardial lipid content. Hypertrophy, documented by an increase in H/W, was evident in aged hearts in our experiments and is consistent with results from other studies performed in mice at 22–24 mo (20). However, we demonstrated preserved systolic and diastolic function in aged isolated working hearts, suggesting that the decrease in cardiac output in vivo is related to elevated systemic vascular resistance or alteration in neurohormonal stimulation and not to intrinsic inotropy.

In contrast to the younger hearts, the aged mouse hearts respond to insulin by increasing cardiac function. The variable inotropic responses of different isolated heart preparations to insulin have been well documented in the literature (1, 28). These have been attributed in part to differences in Ca²⁺ concentration, transport, or Ca²⁺ sensitivity in particular experimental preparations. In the present study, insulin prompted the inotropic response in the aged hearts, with no concomitant increase in CF or MV̇o₂. The dramatic increase in Fc through unlabeled sources in conjunction with the increase in cardiac energy efficiency suggests that the inotropic response has a metabolic basis. The isotopomer analyses in the NMR experiment allow discrimination in the glutamate spectrum for only three labeled substrates. Accordingly, we were not able to label glucose simultaneously with FFA, lactate, and acetoacetate in these experiments. Therefore, we could did not directly measure glucose Fc or flux. Also, we did not measure glucose uptake, often considered a measure of insulin sensitivity. Our prior studies (13) have shown that when the unlabeled fraction increases along with metabolic efficiency, both glucose and glycogen-derived glucose-6-phosphate comprise the majority of this component. Because of the lower ATP-to-O ratio associated with fatty acid oxidation, an increased Fc from endogenous triglycerides, the only other potential source, would decrease metabolic efficiency (11). Thus the data suggest that insulin promotion of oxidation of substrate via pyruvate dehydrogenase is responsible for the inotropic stimulation in the aged hearts. This insulin response is limited in the younger hearts, which maintain a relatively high Fc from fatty acids and ketones. These data suggest that the aged heart retains the ability to respond to immediate stress at least under fully aerobic conditions. The aged mouse heart has been noted to respond poorly to ischemia and reperfusion compared with the younger heart (20). The duration of the stress response in the aged heart may be limited by depletion of endogenous substrate, presumably glycogen.

We also identified a reduction in the anaplerotic response to insulin in aged hearts. Extensive literature review could not reveal another report regarding the specifics of insulin influence on anaplerotic pathways in heart. Therefore, any comment regarding the mechanism responsible for the difference between control and aged hearts remains speculative. Anaplerosis refers to carbon entry into the CAC via molecules other than acetyl-CoA. (24). Anaplerotic pathways include pyruvate carboxylation and amino acid transamination to CAC intermediates, among others (16). Recent studies have shown that pyruvate entry into the CAC via malate compensates for reduced carbon entry when fatty acid oxidation is impaired in hypertrophied heart induced by aortic constriction (25). Studies performed in vivo showed that pyruvate entering the CAC via carboxylation preserves cardiac function after ischemia and reperfusion (24). The present study was not designed to pinpoint the specific anaplerotic pathways that are impaired with aging. We did examine pyruvate carboxylase content but noted no difference between control and aged mice. Carbon entry to the CAC via anaplerosis reduces the energy efficiency of oxidative phosphorylation as anaplerosis bypasses ATP or NADH-producing reactions. Suppression of anaplerosis may contribute to the relatively high rate of energy efficiency in the aged heart. A reduction in anaplerosis could also represent a mode of amino acid preservation for protein synthesis in aged hearts, which are subject to disturbances in protein accumulation (7).

In summary, we show that fatty acid and ketone oxidation are impaired in the aging mouse heart. Despite hypertrophy, the aging mouse heart maintains function at rest by accessing a variety of substrates. Depression of fatty acid Fc and flux appears to be promoted by a decrease in PPARα, a transcriptional activator of PDK4, which is in turn suppressed. Regulation of PPARα during aging in mice may be influenced by systemic changes in lipid and thyroid hormone homeostasis. Insulin does stimulate an inotropic response in aged hearts, supported by increased oxidation of glycolytic substrate. However, carbon contribution to the CAC through fatty acid oxidation and insulin-stimulated anaplerosis is impaired in aging mouse heart. These findings suggest that the aging heart has a limited capability to maintain CAC intermediates during prolonged stress, and may explain in part previous findings of diminished tolerance to insult.

GRANTS

This study was supported by National Institute on Aging Grant R21-AG-0033815 to M. A. Portman, Seattle Mouse Metabolic Phenotyping Center (U24-DK-076126), and Pacific Northwest Laboratories.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).