Long-term Dietary n3 Fatty Acid Prevents Aging-related Cardiac Diastolic and Vascular Dysfunction
Abstract Aims: The prevalence of left ventricular (LV) diastolic and vascular dysfunction increases with age, eventually leading to heart failure with preserved ejection fraction (HFpEF). A preventive strategy is an unmet medical need. We and others reported previously on the beneficial effects of omega-3 fatty acid alpha linolenic acid (ALA) on cardiovascular disorders in animal models and translational studies. We now investigate whether long-term dietary ALA could prevent LV diastolic dysfunction and vascular aging in a murine model. Methods and Results: Wild-type C57BL/6J mice were fed a chow or ALA diet for 12 months, starting at 6 months of age. Here, we show that aged (~18 months) mice recapitulate major hallmarks of HFpEF, including LV diastolic dysfunction with preserved ejection fraction, impaired vascular function, cardiac fibrosis, arterial stiffening and inflammation, as well as elevated B-type natriuretic peptide (BNP). Long-term ALA supplementation upregulated the mitochondrial tricarboxylic acid enzyme Idh2 and the antioxidant enzymes SOD1 and Gpx1. It also counteracted inflammation and ECM remodeling by regulating NF-κB and in turn significantly reducing fibrosis biomarkers MMP-2/TGF-β in both cardiac and vascular tissues obtained from aged mice. These mechanisms emphasize the beneficial preventive effects of ALA on LV diastolic dysfunction, impaired vasorelaxation, cardiac fibrosis, inflammation and arterial stiffening in aged mice, considered as hallmarks of HFpEF. Conclusions: We provide evidence and mechanistic insight on how long-term ALA supplementation is a successful strategy to prevent the development of age-related diastolic and vascular dysfunction.
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- Mice were anesthetized with 1.5–2% isoflurane and subjected to conventional transthoracic echocardiography and Doppler analyses using a VisualSonics Vevo 3100 system equipped with an MX250 (13–24 MHz) probe. Standard M-mode images were obtained from parasternal short- and long-axis views of the left ventricle (LV) at the level of the midpapillary muscle. Anterior and posterior end-diastolic and end-systolic LV wall thickness was measured to calculate EF. Doppler signals of mitral inflow and myocardial tissue movement were obtained to calculate the ratio of peak early (E) to late (A) diastolic transmitral flow velocities (E/A) to assess diastolic function. Both the echocardiography operator and analyzer were blinded to the experimental groups. - Volume pressure recording (VPR) sensors and occlusion cuffs, controlled by a programmed electrosphygmomanometer system (Coda, Kent Scientific), were placed at the distal end of the tail for recording systolic and diastolic blood pressures. - Carotid artery pulse wave velocity was performed using Vevo 3100 system (VisualSonics) equipped with an MS550D 40-MHz linear array transducer (VisualSonics). - Animals were euthanized by CO2 inhalation and whole blood was collected by cardiac puncture using EDTA and centrifuged at 125 g for 8 min for plasma isolation. - Ex vivo Force Tension Myography The murine aortic sections were mounted and connected to an isometric force transducer (PowerLab 8/30 and LabChart v7.2.5, AD Instruments, Inc.) in Krebs-Ringer bicarbonate solution. Concentration–response curves were obtained in a cumulative fashion in response to increasing concentrations of acetylcholine (Sigma-Aldrich), sodium nitroprusside (Sigma-Aldrich), and phenylephrine (Sigma-Aldrich). - Histological analyses of murine hearts fixed in formalin and embedded in paraffin were performed by Masson's trichrome staining. - Quantitative RT-PCR analyses were performed using RNA extracted from murine hearts and aortas with TRI Reagent® (Sigma-Aldrich). - Immunoblot analyses of protein expression were performed using primary antibodies (1:1000) against MMP-2, COX-2, Idh2, phospho-eNOSSer1177, and total eNOS (all from Cell Signaling Technology) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Merck Millipore, Finland). The membranes were then incubated with corresponding HRP-labeled secondary antibodies (1:2000, Southern Biotechnology, USA). Immunoblots were analyzed by chemiluminescence using an Amersham Imager 600 (GE Healthcare Europe GmbH, Glattbrugg, Switzerland). - Plasma BNP levels were quantified in accordance with the manufacturer’s instruction (Sigma-Aldrich). - MDA levels in cardiac and aortic tissue homogenates were quantitated by TBARS assay. - Tissue nitrate levels were measured by Griess method27 in cardiac and aortic tissue homogenates using 2,3-diaminonaphthalene (DAN) reagent provided by nitrate/nitrite fluorometric assay kit (Cayman Chemical, 780051).