Multiple common comorbidities produce left ventricular diastolic dysfunction associated with coronary microvascular dysfunction, oxidative stress, and myocardial stiffening

Abstract

Aims: More than 50% of patients with heart failure have preserved ejection fraction characterized by diastolic dysfunction. The prevalance of diastolic dysfunction is higher in females and associates with multiple comorbidities such as hypertension (HT), obesity, hypercholesterolemia (HC), and diabetes mellitus (DM). Although its pathophysiology remains incompletely understood, it has been proposed that these comorbidities induce systemic inflammation, coronary microvascular dysfunction, and oxidative stress, leading to myocardial fibrosis, myocyte stiffening and, ultimately, diastolic dysfunction. Here, we tested this hypothesis in a swine model chronically exposed to three common comorbidities.

Methods and results: DM (induced by streptozotocin), HC (produced by high fat diet), and HT (resulting from renal artery embolization), were produced in 10 female swine, which were followed for 6 months. Eight female healthy swine on normal pig-chow served as controls. The DM + HC + HT group showed hyperglycemia, HC, hypertriglyceridemia, renal dysfunction and HT, which were associated with systemic inflammation. Myocardial superoxide production was markedly increased, due to increased NOX activity and eNOS uncoupling, and associated with reduced NO production, and impaired coronary small artery endothelium-dependent vasodilation. These abnormalities were accompanied by increased myocardial collagen content, reduced capillary/fiber ratio, and elevated passive cardiomyocyte stiffness, resulting in an increased left ventricular end-diastolic stiffness (measured by pressure-volume catheter) and a trend towards a reduced E/A ratio (measured by cardiac MRI), while ejection fraction was maintained.

Conclusions: The combination of three common comorbidities leads to systemic inflammation, myocardial oxidative stress, and coronary microvascular dysfunction, which associate with myocardial stiffening and LV diastolic dysfunction with preserved ejection fraction.

Figure 1

Mean arterial pressure (MAP), measured in resting awake state, was increased over time in DM + HC + HT as compared to healthy control swine from our laboratory (unpublished data) performed in the same time period (A). Representative PAS stained sections of the top part of the left kidney of control and DM + HC + HT swine at a magnification of 200×. Scale bar = 50 μm (B). Increased tubulo-interstitial damage in kidney sections of DM + HC + HT swine compared to control healthy swine (C). Total tubulo-interstitial damage score was calculated by summing the scores for peritubular inflammatory infiltrate (not shown), interstitial fibrosis (D), atrophy (E) and dilatation (F) (Control N = 8, DM + HC + HT N = 11). *P < 0.05, ^#P = 0.06.

Figure 2

Concentration response curves to bradykinin (BK, A) and the NO-donor S-nitroso-N-acetylpenicillamine (SNAP, B) in small arteries isolated from DM + HC + HT and healthy control hearts. *P < 0.05 DM + HC + HT vs. Control by 2-way ANOVA.

Figure 3

NO production was decreased in the LV subendocardium of DM + HC + HT (N = 10) vs. Controls (N = 8) (A). However, myocardial eNOS expression was increased in DM + HC + HT (B), as was the monomer/dimer (MoDi) ratio (C), suggestive of eNOS uncoupling. Phosphorylation of eNOS (peNOS) was also significantly increased in DM + HC + HT (D) (N = 10) vs. Control (N = 8). Superoxide generation was increased in the LV subendocardium of DM + HC + HT vs. Controls and was suppressed by L-NAME and VAS2870 (E). Upon NADPH oxidase stimulation, the superoxide anion production was dramatically increased (F), which was inhibited by VAS2870 but not by L-NAME treatment. *P < 0.05 DM + HC + HT vs. Control; ^†P < 0.05 vs. corresponding basal.

Figure 4

Examples of histological staining for quantification of collagen deposition (Picrosirius Red A, D), myocyte size (Gomori B, E), and capillary density (Lectin C, F) in the LV subendocardium in Control (CON), and DM + HC + HT animals.

Figure 5

Increased collagen deposition (A) but no myocyte hypertrophy (B) was recorded in LV subendocardium of the DM + HC + HT animals. Capillary density was similar between groups (C), however the capillary-to-fiber ratio was significantly reduced in DM + HC + HT (D). *P < 0.05 DM + HC + HT (N = 10) vs. Control (N = 8).

Figure 6

Increased maximal (F_max, A) and passive force (F_pas, B) were seen in DM + HC + HT as compared to Controls. Cardiomyocyte data were averaged for all measured cells and group averages are shown. Titin N2BA/N2B isoforms ratio was significantly decreased in DM + HC + HT animals (C). Typical examples of E and A waves in Control (D) and DM + HC + HT (E) animals and pressure–volume relationships (G) are presented. The early to late filling (E/A) ratio tended to be lower in the DM + HC + HT animals (F). End-diastolic elastance (slope of EDPVR, sEDPVR) was significantly increased (H) while a trend towards significance in the end-systolic elastance (slope of ESPVR, sESPVR) was recorded (I), *P < 0.05, ^#P = 0.10 DM + HC + HT vs. Control.

Figure 7

Relation between left atrial pressure (LAP) and cardiac index (CI) at rest and during treadmill exercise in chronically instrumented Control (N = 4; 97 ± 6 kg, white circles) and DM + HC + HT (N = 4; 94 ± 7 kg, black circles) swine. *P < 0.05 DM + HC + HT vs. Control.

Figure 8

In a large animal, chronic exposure to multiple common comorbidities results in systemic inflammation, endothelium-dependent coronary artery dysfunction, capillary rarefaction, oxidative stress, and perturbed nitric oxide production, which are associated with increased myocardial fibrosis and passive cardiomyocyte stiffness, resulting in LV diastolic dysfunction. Adapted with permission from Ter Maaten et al. The findings of the present study that are in agreement with the hypothesis are presented in bold.