Lard Intake Reduced Tetralinoleoyl Cardiolipin and Attenuated the Cardioprotective Effects of Exercise in Mice With Pressure Overload-Induced Heart Failure

Abstract

Background

Exercise-based cardiac rehabilitation is effective for improving the prognosis of patients with heart failure (HF), but little is known about its interactions with dietary components. We investigated the impacts of dietary fat intake and its source on the cardioprotective effects of exercise in mice with HF.

Methods and Results

Endurance exercise and diets with different fat composition, including a normal diet (NE: 11% fat), a soybean oil-rich diet (SE: 32% fat) and a lard-rich diet (LE: 32% fat), were provided for 4 weeks to mice with HF induced by transverse aortic constriction. Exercise improved left ventricular systolic function and suppressed cardiac hypertrophy in the NE and SE groups, but these effects were attenuated in the LE group. The LE group exhibited a reduction in linoleic acid content within mitochondrial phospholipids such as phosphatidylcholine and phosphatidylethanolamine, concurrent with the promotion of cardiolipin remodeling pathways. This resulted in a marked reduction in tetralinoleoyl cardiolipin (L₄CL), which is crucial for maintaining mitochondrial membrane function. This was supported by the finding that assembly of mitochondrial respiratory chain supercomplexes was partially impaired in the LE group.

Conclusions

Lard intake may attenuate the exercise-induced cardioprotective effects against HF by reducing myocardial L₄CL levels and impairing mitochondrial supercomplex assembly involved in mitochondrial respiratory capacity.

Central Figure.

Heart failure (HF) is a serious global health concern, and its prevalence is increasing due to aging populations and the rising incidence of lifestyle-related diseases such as obesity and diabetes.^1–3 HF progressively worsens and is related to low quality of life and increased healthcare costs for patients, making comprehensive and multidisciplinary care essential.

Exercise-based cardiac rehabilitation is a well-established non-pharmacological therapy.⁴ It not only improves physical deconditioning, such as exercise intolerance and muscle weakness, but also myocardial contractility and energy metabolism,^5,6 including improvements in cardiac energetics, antioxidant capacity, mitochondrial function, and promotion of mitophagy.^7–10

Nutritional therapy has also gained consensus as a highly recommended treatment option that is expected to enhance the effectiveness of other therapeutic interventions.^11,12 Interestingly, several animal studies have reported the cardioprotective effects of a high-fat diet (HFD), with the beneficial effects thought to be mediated by enhanced myocardial fatty acid oxidation and alterations in mitochondrial phospholipid profiles associated with mitochondrial function.^13–15 Importantly, many of the pathways regulated by exposure to HFD are also molecular targets of exercise,^7,9,13,15 suggesting potential interactions in vivo. However, the influence of dietary fat composition on the cardioprotective effects of exercise remains unknown, and furthermore, whether a HFD is beneficial for a failing heart appears to depend on the source of fat consumed; that is, whether it is of plant or animal origin.^16,17

Hence, in this study we explored how soybean oil and lard, the most frequently consumed plant and animal fats, respectively, affected the benefits of exercise in mice with HF induced by transverse aortic constriction (TAC). Clarifying these effects may provide novel insights into nutritional management during cardiac rehabilitation for HF.

Methods

Animals

The 5-week-old male C57BL/6 mice were obtained from Japan SLC, Inc. (Shizuoka, Japan) and kept in a climate-controlled room (22± 2℃) under a 12:12-hour light/dark cycle with ad libitum access to standard chow (MF, Oriental Yeast, Tokyo, Japan) and water before all experiments. All animal experiments were approved by the Animal Experimentation Committee of Tokushima University School of Medicine (Approval No. T2021-104) and conducted in accordance with the guidelines of the Animal Care and Use Committee of Tokushima University School of Medicine. All procedures complied with the NIH Guide for the Care and Use of Laboratory Animals.

Transverse Aortic Constriction (TAC)

TAC was performed as previously described.¹⁸ At 8-weeks old, the mice were anesthetized using 1.5–2% isoflurane. After a midline sternotomy extending from the suprasternal notch to the second rib, a 7-0 silk ligature was placed around the transverse aorta between the innominate artery and the left common carotid artery and tied around a blunt 27-gauge needle to ensure consistent constriction. Following ligation, the needle was promptly removed. The sham operation was performed using the same surgical procedure but without placement or tightening of the ligature around the vessel.

Experimental Design

According to research on the beneficial effects of exercise in HF models,^19,20 in the preliminary test (Experiment 1) to determine the appropriate exercise intensity, the following groups were established: sham sedentary (SS), TAC sedentary (TS), TAC low-intensity training (TL), and TAC high-intensity training (TH). All mice were provided with the standard chow. Treadmill training was performed at either low intensity (12 m/min for 30 min/day, 3 days/week) or high intensity (12 m/min for 60 min/day, 5 days/week) for 8 weeks.

In the main test (Experiment 2) to examine the effects of diet on the efficacy of exercise, the experimental groups included the following: normal diet + sedentary (NS), normal diet + exercise (NE), soybean oil diet + exercise (SE), and lard diet + exercise (LE). The normal diet consisted of standard chow supplemented with 10% dextrin (Oriental Yeast). In the test diets, dextrin was replaced with either soybean oil (Fujifilm Wako Pure Chemical, Osaka, Japan) or lard (Marin Food, Osaka, Japan), and referred to as the soybean oil diet and lard diet, respectively. Detailed compositions of the diets are presented in the Table. In addition to the findings from Experiment 1, the intervention period for Experiment 2 was set at 4 weeks to minimize the adverse effects associated with long-term HFD feeding.²¹ Treadmill training was conducted at 12 m/min for 30 min/day, 3 days/week, for 4 weeks.

Table.

Composition of Experimental Diets

	Normal diet	Soybean oil diet (g/100 g)	Lard diet
Protein	20.8	20.8	20.8
Fat	4.6	14.6	14.6
Carbohydrate	59.8	49.8	49.8
Other	14.8	14.8	14.8
		(% of total FA)
C16:0	15.2	12.1	21.8
C16:1	1.0	0.4	2.0
C18:0	2.8	3.8	10.5
C18:1	26.3	24.5	37.9
C18:2	49.0	52.4	22.0
C18:3	3.8	5.7	1.5
Other	1.9	1.1	4.3

The nutrient composition of the experimental diets is shown. FA composition is presented as estimated values expressed as percentages of total FA. FA, fatty acid.

In both experiments, mice were evenly assigned to each group based on fractional shortening (FS), an echocardiographic parameter of left ventricular (LV) systolic function measured 1 week after TAC surgery. After the intervention, all mice were humanely killed using deep isoflurane anesthesia (≥5%) followed by exsanguination via blood collection from the inferior vena cava for heart tissue collection.

Echocardiographic Analysis

Mice were anesthetized using 1.5–2% isoflurane, and LV systolic function was then evaluated using a MyLab Sigma VET ultrasound unit (Esaote, Liguria, Italy) equipped with a 22.0 MHz probe (SL3116, Esaote). M-mode images were obtained in the short-axis view at the papillary muscle level. LV end-diastolic internal diameter (LVIDd), LV end-systolic internal diameter (LVIDs), and LV posterior wall thickness (PWT) were evaluated, and FS was calculated as: FS (%) = (LVIDd − LVIDs) / LVIDd × 100. For each animal, measurements were averaged from 3 independent images.

Real-Time Quantitative Polymerase Chain Reaction (PCR)

Total RNA was extracted from heart tissue using the ISOSPIN Cell & Tissue RNA kit (NIPPON GENE, Tokyo, Japan) according to the manufacturer’s instructions. cDNA was synthesized from 1 µg of total RNA using the ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan). Real-time quantitative PCR was conducted using Fast SYBR Green PCR Master Mix and a real-time PCR system (Thermo Fisher Scientific, MA, USA). To confirm the presence of a single PCR product in all reactions, except for the negative controls, amplification products were evaluated with melting curve analysis. mRNA levels were measured using the ∆∆Ct method and normalized to Gapdh as the housekeeping gene. The primer sequences used are presented in Supplementary Table 1.

Western Blot

Total protein was extracted from heart tissue using the RIPA buffer (Fujifilm Wako Pure Chemical), which contained protease and phosphatase inhibitors (Nacalai Tesque, Kyoto, Japan). Protein samples were dissolved in sample buffer containing 5% 2-mercaptoethanol followed by SDS-PAGE (5–15 μg protein/lane). The separated proteins were then transferred to polyvinylidene difluoride (PVDF) transfer membranes (Immobilon-P, Millipore, MA, USA) through electrophoresis and incubated with appropriate primary antibodies, followed by HRP-conjugated secondary antibodies. Details of the primary and secondary antibodies are provided in Supplementary Table 2. Signals were detected using Chemi-Lumi One Super and Chemi- Lumi One L (Nacalai Tesque) and visualized using a luminescent image analyzer (LAS-3000UVmini, Fujifilm, Tokyo, Japan). Data quantification was conducted using the Multi Gauge software ver. 3.0 (Fujifilm).

Blue Native PAGE (BN-PAGE)

For BN-PAGE, mitochondrial proteins (20 μg) were solubilized in 1×Sample Buffer (20 mM HEPES, 150 mM KCl, 100 mM sucrose, and 1 mM EDTA) supplemented with 8 g of digitonin/g of mitochondrial protein for 20 min on ice. The lysate then underwent centrifugation at 20,000×g for 10 min at 4℃, and the supernatant was retrieved and supplemented with EzApply Native (ATTO, Tokyo, Japan). Samples were loaded onto 3–10% u-PAGEL H gels (ATTO). Electrophoresis was conducted at 150 V for 90 min with EzRun BlueNative (ATTO). After electrophoresis, the gels were transferred to PVDF membranes, which were fixed and destained with 8% acetic acid and 50% methanol for 15 min. The PVDF membranes were then processed using standard western blot procedures.

Mitochondrial Isolation

Mitochondria were isolated using the Mitochondria Isolation Kit for Tissue and Cultured Cells (Abcam) according to the manufacturer’s protocol. Briefly, heart tissue was minced in 10 volumes of mitochondria isolation buffer and homogenized using a Dounce tissue grinder with 15 strokes. To remove cellular debris, the homogenate underwent centrifugation at 600×g for 10 min at 4℃, and the supernatant was retrieved and further centrifuged at 7,000×g for 10 min at 4℃ to pellet the mitochondria. The mitochondrial pellet was then carefully resuspended in Storage Buffer. The isolated mitochondria were either used immediately or pelleted again at 7,000×g for 10 min at 4℃ and stored at −80℃.

LC-MS

Measurement of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cardiolipin (CL) levels was performed as previously described.²² After adding the corresponding internal standards, total lipids were extracted overnight from isolated heart mitochondria (75 µg) using 1 mL of chloroform/methanol (2 : 1, v/v) that contained 0.2 mg/mL butylated hydroxytoluene. The extracts were evaporated under vacuum till they dried, and the samples were reconstituted in solvent mixtures according to the lipid to be analyzed: for PC and PE measurement, samples were reconstituted in acetonitrile/isopropanol/H₂O (65 : 30:5, v/v/v) and for CL measurement, samples were reconstituted in acetonitrile/isopropanol/H₂O (29 : 55.8 : 15.2, v/v/v). Following ultrafiltration, the sample was transferred into the LC-MS system. PC, PE, and CL levels were measured using an LC-MS-8040 triple quadrupole mass spectrometer (Shimadzu, Kyoto, Japan) equipped with an electrospray ionization probe, an LC-30AD binary pump (Shimadzu), a SIL-30AC autosampler (Shimadzu), and a CTO-20AC column oven (Shimadzu). High-performance liquid chromatography was conducted using an Accucore RP-MS column (2.6 μm, 2.1×50 mm; Thermo Fisher Scientific). The mobile phase comprised (A) H₂O/acetonitrile (60 : 40, v/v) and (B) isopropanol/acetonitrile (90 : 10, v/v), with both being supplemented with 10 mM ammonium formate and 0.1% formic acid. The flow rate was set to 0.4 mL/min, and the gradient program for PC and PE evaluation was as follows: 10% B at 0 min, 10% B at 2 min, 100% B at 15 min, 10% B at 15.01 min, and 10% B at 20 min. Meanwhile, the gradient program for CL evaluation was as follows: 40% B at 0 min, 40% B at 2 min, 100% B at 15 min, 100% B at 25 min, and 40% B at 30 min. For MS/MS analysis, the nebulizer gas flow, drying gas flow, desolvation line temperature, heat-block temperature, and collision-induced dissociation gas pressure were set to 3.0 L/min, 15.0 L/min, 250℃, 400℃, and 230 kPa, respectively. The peak area of each individual phospholipid species was normalized against the peak area of the internal standard. The relative abundance of each phospholipid species was confirmed by normalizing its peak area against the total peak area within its respective phospholipid class (expressed as percentage). For the CL species, the peak area was normalized against the peak area of CL56:0 and the mitochondrial protein content.

Statistical Analysis

Data are reported as mean ± standard error of the mean. The two-tailed unpaired t-test was used for two-group comparisons and for multiple comparisons, one-way analysis of variance followed by Tukey’s tests. All analyses were performed using GraphPad Prism 8 software (Graphpad Software, CA, USA). P values <0.05 were considered statistically significant.

Results

Exercise Exerts Cardioprotective Effects in TAC Mice

Experiment 1 was conducted to confirm the appropriate training intensity in our TAC-induced HF model. At week 4, FS in all TAC groups was significantly lower than that in the SS group (Supplementary Figure A,B). Among the TAC groups, FS was significantly higher in both the TL and TH groups compared to the TS group. Although the LVIDd did not significantly differ among the groups, the LVIDs was significantly higher in the TS group compared to the other groups. Additionally, the PWT was significantly higher in both the TS and TL groups compared to the SS group, but this increase was attenuated in the TH group. At week 8, the improvement in FS was maintained in both exercise groups compared to the TS group (Supplementary Figure A,C). These results suggested that both low- and high-intensity treadmill training was cardioprotective in TAC mice.

Effect of Lard Intake on the Beneficial Effects of Exercise on Cardiac Function

In Experiment 2, we examined the effects of simultaneous dietary fat and exercise interventions. After 4 weeks, the FS in the NE and SE groups was significantly higher than that in the NS group (Figure 1A,B). In contrast, FS did not increase in the lard diet+exercise (LE) group. LVIDd and PWT did not significantly differ among the groups, but LVIDs was significantly lower in the SE group compared to the NS group.

Figure 1.

Effects of dietary fat composition on the beneficial effects of exercise in TAC mice. (A) Representative echocardiographic images. (B) Echocardiographic analysis of fractional shortening (FS), left ventricular end-diastolic internal diameter (LVIDd), left ventricular end-systolic internal diameter (LVIDs), and left ventricular posterior wall thickness (PWT). (C) Heart weight normalized to the tibia length. (D) Relative myocardial mRNA expression of Anp and Bnp. Data are shown as mean±standard error of the mean (n=6–12). Statistical analysis was conducted using one-way analysis of variance followed by Tukey’s tests. *P<0.05, **P<0.01, ***P<0.001.

Furthermore, heart weights in the NE and SE groups were significantly lower than that in the NS group, but no such reduction was noted in the LE group (Figure 1C), suggesting more advanced hypertrophic remodeling in the LE group compared with the NE and SE groups. In contrast, the mRNA expressions of atrial and brain natriuretic peptides were significantly lower in the NE, SE, and LE groups compared to the NS group, indicating that exercise still had a positive beneficial effect regardless of dietary fat composition (Figure 1D). These results suggested that lard intake had partially attenuated the cardioprotective effects of exercise in TAC mice.

Effect of Increased Fat Intake on Cardiac Fatty Acid Oxidation

To assess the effects of dietary fat composition on myocardial fatty acid oxidation, we evaluated the mRNA expression of relevant genes. Our results revealed that the expression of fatty acid translocase (CD36), carnitine palmitoyl transferase 1B (CPT1B), and acyl-CoA dehydrogenase medium-chain (ACADM) were higher in the SE and LE groups compared to the NS and NE groups (Figure 2A). In addition, these protein expressions tended to be higher in the SE and LE groups compared to the NS and NE groups (Figure 2B,C), suggesting that fat intake may have enhanced fatty acid oxidation in the myocardium. The findings suggested that a reduction in myocardial fatty acid oxidation was not responsible for the attenuation of the cardioprotective effects of exercise in the LE group.

Figure 2.

Effects of dietary fat composition on fatty acid oxidation in the myocardium. (A) Relative myocardial mRNA expression of Cd36, Cpt1b, and Acadm. (B) Western blot analysis of myocardial CD36, CPT1B, and ACADM. (C) Quantification of western blot band intensities. GAPDH was used as the loading control. Data are shown as mean ± standard error of the mean (n=6–7). Statistical analysis was conducted using one-way analysis of variance followed by Tukey’s tests. *P<0.05, **P<0.01, ***P<0.001.

Effect of Lard Intake on Phospholipid Profiles in the Myocardium

In the myocardial mitochondria of the LE group, the proportion of stearic acid (C18:0) in both PC and PE was higher than in the other groups, and the proportion of oleic acid (C18:1) was higher than that in the SE group (Figure 3A,B). Additionally, the proportion of linoleic acid (C18:2) in the LE group was significantly lower than that in the other groups.

Figure 3.

Effect of dietary fat composition on myocardial phospholipids. (A) Ratios of stearic acid (C18:0), oleic acid (C18:1), and linoleic acid (C18:2) in mitochondrial phosphatidylcholine (PC). (B) Ratios of stearic acid (C18:0), oleic acid (C18:1), and linoleic acid (C18:2) in mitochondrial phosphatidylethanolamine (PE). (C) Relative myocardial mRNA expression of Cds2 and Crls1. (D) Relative myocardial mRNA expression of Lclat1 and Tafazzin. Data are shown as mean ± standard error of the mean (n=5–8). Statistical analysis was conducted using one-way analysis of variance followed by Tukey’s tests. *P<0.05, **P<0.01, ***P<0.001.

We then evaluated the myocardial mRNA expression of genes involved in the synthesis and remodeling of CL, which is a phospholipid specific to the mitochondrial membrane. Our findings revealed that the expression of CDP-diacylglycerol synthase 2 and cardiolipin synthase 1, which are responsible for CL synthesis, did not differ significantly among the groups (Figure 3C). In contrast, the expression of lysocardiolipin acyltransferase 1 and phospholipid-lysophospholipid transacylase (TAFAZZIN), which are involved in CL remodeling, was higher in the SE and LE groups compared to the NS and NE groups (Figure 3D). These findings indicated that dietary fat induced alterations in mitochondrial phospholipid composition, and along with enhanced CL remodeling, may have contributed to changes in the fatty acid composition of CL.

Effect of Lard Intake on Tetralinoleoyl CL Levels

We then evaluated the fatty acid composition of myocardial CL to determine whether the alterations in the phospholipid fatty acid composition in the LE group affected the CL profile. Levels of CL72:4, CL72:5, and CL72:6, which contain stearic acid and/or oleic acid in their acyl chains, were significantly higher than in the other groups, while CL72:7 levels were slightly lower (Figure 4). The predominant CL species in the heart is tetralinoleoyl CL (L₄CL; CL72:8), which contains 4 linoleic acid chains and plays a key role in maintaining mitochondrial structure and function.^23,24 Notably, CL72:8 levels were markedly lower in the LE group than in the other groups. These findings indicated that the reduction in L₄CL levels may have contributed to the impaired mitochondrial respiratory capacity in the LE group.

Figure 4.

Fatty acid composition of individual cardiolipin species (C72:4, C72:5, C72:6, C72:7, C72:8) in cardiac mitochondria. Data are shown as mean ± standard error of the mean (n=5–8). Statistical analysis was conducted using one-way analysis of variance followed by Tukey’s tests. *P<0.05, **P<0.01, ***P<0.001.

Effect of Lard Intake on Mitochondrial Supercomplex Assembly

Because a reduction in L₄CL levels impair mitochondrial supercomplex assembly,^25,26 we analyzed the assembly of mitochondrial supercomplexes in the myocardium. The protein expression levels of individual mitochondrial respiratory chain complexes did not differ significantly in the LE group (Figure 5A,B). However, in the LE group, levels of the highest molecular weight supercomplex (Band No. 1) were significantly lower than that in the SE group (Figure 5C,D), suggesting impairment in the assembly of supercomplexes comprising Complex I and Complex III (Figure 5C).

Figure 5.

Effects of dietary fat composition on mitochondrial supercomplexes in cardiac mitochondria. (A) Western blot analysis of proteins involved in mitochondrial oxidative phosphorylation in the myocardium. (B) Quantification of western blot band intensities. GAPDH was used as the loading control. (C) BN-PAGE and western blot analysis of OXPHOS, NDUFS1, UQCRC2, and COX4 in cardiac mitochondria. (D) Quantification of western blot supercomplexes band intensities. Tim23 was used as the loading control. Data are shown as mean ± standard error of the mean (n=6–7). Statistical analysis was conducted using one-way analysis of variance followed by Tukey’s tests or a two-tailed unpaired t test. *P<0.05, ***P<0.001.

Discussion

The results from this study showed that the cardioprotective effects of endurance exercise were influenced by differences in the dietary fat source, as evidenced by lard intake partially attenuating the beneficial effects of exercise. Our findings also demonstrated that decreased levels of myocardial L₄CL, which impairs mitochondrial supercomplex assembly, may have contributed to the attenuation of the cardioprotective effects of exercise following lard intake.

CL is a dimeric glycerophospholipid that is mostly located in the inner mitochondrial membrane. It is important for the activation and stabilization of mitochondrial proteins, including those involved in oxidative phosphorylation and the electron transport chain.^23,24 Additionally, CL plays a crucial role in efficient electron transfer and ATP synthesis by maintaining the structural integrity of mitochondrial supercomplexes.^25–27 In the myocardium, L₄CL is a particularly essential molecular species for effectively exerting physiological functions, accounting for approximately 80% of the total myocardial CL content. In cardiomyocyte-specific TAFAZZIN knockout mice, loss of mitochondrial phospholipid-lysophospholipid acyltransferase function impairs CL remodeling and maturation, resulting in decreased total CL and L₄CL. This disruption of cardiolipin homeostasis has been shown to inhibit the assembly of mitochondrial supercomplex, leading to reduced mitochondrial respiratory capacity.²⁸ Although mitochondrial respiration could not be assessed in the present study, the findings suggested that the reduction in L₄CL levels in the LE group may have contributed to the poor exercise responsiveness via impaired mitochondrial respiratory function. Although increased oxidative stress and inflammation due to lard intake may also be involved,²⁹ we further propose this cardiolipin-related mechanism as a novel pathway contributing to the attenuation of exercise-induced cardioprotection following a lard-rich diet.

In normal mammals, the amount of dietary linoleic acid strongly contributes to maintaining L₄CL homeostasis.^16,30,31 However, the net linoleic acid content of the current lard-rich diet was higher than that of the normal diet fed to the NE group, which indicates that the L₄CL level in the LE group decreased regardless of the relatively high linoleic acid intake. In the heart, CL is initially synthesized as nascent CL containing multiple fatty acids, followed by the replacement of these fatty acid chains primarily with linoleic acid (C18:2), resulting in functionally mature CL. In this remodeling pathway, TAFAZZIN catalyzes the incorporation of fatty acids derived from intracellular PC and PE into CL, so the fatty acid composition of membrane phospholipid is directly reflected in the fatty acid chain formation of cardiolipin.³² In the LE group, the fatty acid composition of each phospholipid (PC and PE) contained increased stearic acid (C18:0) and decreased linoleic acid (C18:2). It is important to note that this specific ratio was determined not by the individual fatty acid amounts in the diet, but rather by the relative fatty acid composition. On the other hand, TAFAZZIN mRNA expression levels were elevated in both fat-rich diet groups (SE and LE), a finding that is consistent with previous studies reporting increased TAFAZZIN mRNA expression in mouse livers fed a HFD.³³ Taking all findings together, we conclude that in the failing heart under a lard-rich diet, fat loading enhances CL remodeling while the lard-specific fatty acid composition reduces linoleic acid in mitochondrial phospholipids available for CL remodeling, thereby resulting in a significant decrease in L₄CL levels in the LE group compared to the NE group.

It is noteworthy that not all findings in the LE group were adverse; the phenotypes reflecting worsening HF were limited to some data such as LV dysfunction. One plausible explanation for this is that short-term HFD intake suppresses the progression of HF by promoting myocardial fatty acid oxidation, regardless of the lipid source.^13,21 Indeed, mRNA and protein expressions of CD36, CPT1B, and ACADM, which are important regulators of fatty acid oxidation, remained elevated even in the LE group. These data support the possibility that a lard-rich diet may have been beneficial for myocardial energy metabolism during at least the 4-week intervention, potentially related to the restriction of the negative phenotypes. Given the evidence that the beneficial effects of HFD may not last long, especially when consuming animal fats,²¹ the inhibitory effects of lard intake may become more pronounced with long-term intervention. In contrast, the soybean oil-rich diet, which is plant-based and does not cause L₄CL reduction, may have shown a stronger tendency toward improved cardiac function in the SE group than in the NE group, primarily due to enhanced fatty acid oxidation influencing the phenotype. It has been reported that patients with advanced HF have low linoleic acid intake,³¹ which may potentially contribute to worsening through a reduction in myocardial L₄CL. In this study, we used soybean oil and lard as plant- and animal-based fats, respectively, which are commonly consumed daily. However, n-3 polyunsaturated fatty acids and medium-chain triglycerides, known as functional lipids, have beneficial effects on HF,^34,35 but the significance of differences in fatty acid intake during exercise in HF remains unclear. Further research aimed at clarifying these interactions may contribute to the development of novel nutritional strategies optimized for cardiac rehabilitation in patients with HF.

Study Limitation

The main limitation is the exclusive use of male mice, which may limit the generalizability of the findings. Given reported sex-specific differences in cardiac and metabolic responses to HFD,³⁶ the effects of dietary fat on exercise benefits observed here may differ in females. Future studies including both sexes are needed to confirm the broader applicability of these findings.

Conclusions

Our study revealed that lard intake may attenuate the cardioprotective effects of endurance exercise on the progression of HF by reducing L₄CL levels in myocardial mitochondria. Our findings highlight the impact of differences in dietary fatty acid composition rather than the amount of fat, and provide new insights to nutritional management focused on lipid quality in cardiac rehabilitation, which may help maximize the exercise-induced benefits.

Disclosures

No conflicts of interest, financial or otherwise, are declared by the authors. H.O. is a member of Circulation Reports’ Editorial Team.

IRB Information

All animal experiments were approved by the Animal Experimentation Committee of Tokushima University School of Medicine (Approval No. T2021-104)

Data will be made available upon reasonable request.

Supplementary Files

Supplementary File 1

Supplementary Table 1. Supplementary Table 2. Supplementary Figure.

Funding Statement

Grants: This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI grant number 20H04038 (to H.O.); the Japan Science and Technology Agency (JST) SPRING grant number JPMJSP2113 (to A.K.); and the Research Clusters Program of Tokushima University (to H.O.).