Lactobacillus plantarum probiotic induces Nrf2-mediated antioxidant signaling and eNOS expression resulting in improvement of myocardial diastolic function

Abstract

Yorkshire swine were fed standard diet (n = 7) or standard diet containing applesauce rich in caffeic acid with Lactobacillus plantarum (n = 7) for 3 wk. An ameroid constrictor was next placed around the left coronary circumflex artery, and the dietary regimens were continued. At 14 wk, cardiac function, myocardial perfusion, vascular density, and molecular signaling in ischemic myocardium were evaluated. The L. plantarum-applesauce augmented NF-E2-related factor 2 (Nrf2) in the ischemic myocardium and induced Nrf2-regulated antioxidant enzymes heme oxygenase-1 (HO-1), NADPH dehydrogenase quinone 1 (NQO-1), and thioredoxin reductase (TRXR-1). Improved left ventricular diastolic function and decreased myocardial collagen expression were seen in animals receiving the L. plantarum-applesauce supplements. The expression of endothelial nitric oxide synthase (eNOS) was increased in ischemic myocardial tissue of the treatment group, whereas levels of asymmetric dimethyl arginine (ADMA), hypoxia inducible factor 1α (HIF-1α), and phosphorylated MAPK (pMAPK) were decreased. Collateral-dependent myocardial perfusion was unaffected, whereas arteriolar and capillary densities were reduced as determined by α-smooth muscle cell actin and CD31 immunofluorescence in ischemic myocardial tissue. Dietary supplementation with L. plantarum-applesauce is a safe and effective method of enhancing Nrf2-mediated antioxidant signaling cascade in ischemic myocardium. Although this experimental diet was associated with a reduction in hypoxic stimuli, decreased vascular density, and without any change in collateral-dependent perfusion, the net effect of an increase in antioxidant activity and eNOS expression resulted in improvement in diastolic function.NEW & NOTEWORTHY Colonization of the gut microbiome with certain strains of L. Plantarum has been shown to convert caffeic acid readily available in applesauce to 4-vinyl-catechol, a potent activator of the Nrf2 antioxidant defense pathway. In this exciting study, we show that simple dietary supplementation with L. Plantarum-applesauce-mediated Nrf2 activation supports vascular function, ameliorates myocardial ischemic diastolic dysfunction, and upregulates expression of eNOS.

Keywords: antioxidant; chronic myocardial ischemia; diastolic function; endothelial function; probiotic.

Figure 1.

The Lactobacillus plantarum-caffeic acid diet was associated with significant decline in end-diastolic pressure-volume resistance (A) reflective of enhanced myocardial relaxation and diastolic performance in the treated group. These data were acquired by assessment of multiple indices of hemodynamic performance and extrapolation of pressure-volume loops for each of the control controls (n = 7; males n = 3 and females n = 4) (B) and treatment controls (n = 7; males n = 3 and females n = 4) (C) animal groups. Mann–Whitney t test was used for statistical analysis.

Figure 2.

Lactobacillus plantarum-caffeic acid dietary regimen does not have a significant effect on absolute blood flow to the ischemic myocardial region. Conditions shown are under resting (A) and paced (B). Similarly, the perfusion ratio to ischemic territory vs. nonischemic ventricle were unchanged at basal heart rate (C) and while pacing at 150 beats/min (D) between experimental cohorts (n = 7; males n = 3 and females n = 4) and controls (n = 7; males n = 3 and females n = 4) for each group; P values shown for each panel. Mann–Whitney t test was used for statistical analysis.

Figure 3.

Quantification of ischemic myocardial densities of endothelial α-SMA and CD-31 by immunofluorescence. α-SMA (A) and CD31 (B) endothelial cell densities were significantly less in ischemic myocardial tissue of animals that received the Lactobacillus plantarum-caffeic acid diet. Animals that received the experimental diet showed no significance in isolectin B4 density (C) nor the density of vimentin to be significantly altered between the two groups (D), whereas myocardial collagen expression was significantly reduced. Scale bars for representative images are 100 μm (A and B) and 50 μm (C–E).

Figure 4.

Nrf-2-mediated expression of antioxidant proteins in ischemic myocardium. Significant upregulation of Nrf2 signaling is seen in the Lactobacillus plantarum-caffeic acid cohort (n = 7; males n = 3 and females n = 4). Ischemic myocardial lysates from treated animals exhibit greater expression of Nrf2 (A), as well as important downstream antioxidant enzymes including HO-1 (B), NQO-1 (C), and TRXR-1 (D) with respect to controls (n = 7; males n = 3 and females n = 4). Mann–Whitney t test was used for statistical analysis. HO-1, heme oxygenase 1; NDC, normal-diet control; NQO-1, NADPH dehydrogenase quinone 1; Nrf2, nuclear factor erythroid 2-related factor 2; TRXR-1, thioredoxin reductase.

Figure 5.

Increased Nrf2 activity was associated with increased expression of eNOS (A) and reduced levels of ADMA (B) in the treatment (ND-LP) group (n = 7; males n = 3 and females n = 4) compared with normal-diet control (NDC) group (n = 7; males n = 3 and females n = 4). The treatment group demonstrated decreased expression of HIF-1α (C), suggesting decreased hypoxic stress in ischemic myocardium of the treated (ND-LP) group compared with control (NDC), plausibly due to better oxygenation of the ischemic myocardium. Lastly, decreased p-MAPK (D) suggests reduction in proliferative/angiogenic signaling in ischemic myocardium of ND-LP group (n = 6). There was no significant difference between the groups (E) (NDC: n = 6; males n = 3 and females n = 3 and ND-LP: n = 5; males n = 2 females and n = 3) and with respect to phosphorylation of e-NOS in ischemic myocardial lysates. ADMA, asymmetric dimethyl arginine; eNOS, endothelial nitric oxide synthase; HIF-1α, hypoxia inducible factor-1α; Nrf2, nuclear factor erythroid 2-related factor 2; p-eNOS, phosphorylated endothelial nitric oxide synthase; p-MAPK, phosphorylated mitogen activated protein kinase.

Figure 6.

Activation of Nrf2-mediated antioxidant signaling and therapeutic outcomes. A: treated swine were fed an applesauce diet rich in caffeic acid supplemented with L. plantarum, a source of phenolic acid decarboxylase. This enzyme converts caffeic acid to 4-vinyl-catechol, an Nrf-2 activator. B: under normal conditions, Nrf2 is sequestered by Keap1 in the cytoplasm and proteolytically degraded. Oxidative challenge induces Nrf2 dissociation from Keap2 resulting in nuclear translocation of Nrf2. Nrf2 binds to antioxidant response elements (AREs) in the nucleus inducing the expression of a variety of antioxidant proteins. C: protein expression of ADMA was found to be reduced, whereas eNOS expression was increased, suggesting increased availability of nitric oxide (NO), which may have resulted in improved tissue oxygenation. On the contrary, reduction in HIF-1α and pMAPK signaling suggests inhibition of proliferative and angiogenic stimuli in ischemic myocardium of the treated group, which may have resulted in the lack of collateral vessel formation in the treated group. Image created with BioRender and published with permission. ADMA, asymmetric dimethyl arginine; eNOS, endothelial nitric oxide synthase; GPX-1, glutathione peroxidase 1; HIF-1α, hypoxia inducible factor-1α; HO-1, heme oxygenase 1; Keap1, kelch-like ECH-associated protein 1; NQO-1, NADPH dehydrogenase quinone 1; Nrf2, nuclear factor erythroid 2-related factor 2; pMAPK, phosphorylated mitogen activated protein kinase; TXRX1, thioredoxin reductase. Created with BioRender.com; published with permission.