Ischemic cardiomyopathy following seizure induction by domoic Acid

Abstract

Exposure to the excitotoxin domoic acid (DOM) has been shown to produce cardiac lesions in both clinical and animal studies. We have previously shown that DOM failed to directly affect cardiomyocyte viability and energetics, but the development of this cardiomyopathy has remained unexplained. The present study compared effects of high-level seizure induction obtained by intraperitoneal (2 mg/kg) or intrahippocampal (100 pmol) bolus administration of DOM on development of cardiac pathologies in a rat model. Assessment of cardiac pressure derivatives and coronary flow rates revealed a significant time-dependent decrease in combined left ventricular (LV) systolic and diastolic function at 1, 3, 7, and 14 days after intraperitoneal administration and at 7 and 14 days after intrahippocampal DOM administration. LV dysfunction was matched by a similar time-dependent decrease in mitochondrial respiratory control, associated with increased proton leakage, and in mitochondrial enzyme activities. Microscopic examination of the LV midplane revealed evidence of progressive multifocal ischemic damage within the subendocardial, septal, and papillary regions. Lesions ranged from reversible early damage (vacuolization) to hypercontracture and inflammatory necrosis progressing to fibrotic scarring. Plasma proinflammatory IL-1α, IL-1β, and TNF-α cytokine levels were also increased from 3 days after seizure induction. The observed cardiomyopathies did not differ between intraperitoneal and intrahippocampal groups, providing strong evidence that cardiac damage after DOM exposure is a consequence of a seizure-evoked autonomic response.

Figure 1

Cumulative behavioral seizure scores in rats were recorded over 2 hours after intraperitoneal (black bars; 1, 3, 7, and 14 days) or intrahippocampal (gray bars; 7 and 14 days) administration of domoic acid (DOM). Data are expressed as means ± SEM of 8 separate experiments. ***P < 0.001 versus intraperitoneal saline-treated animals (1 and 14 day); ^†††P < 0.001 versus intraperitoneal intrahippocampal saline-treated animals (1 and 14 day). S, saline.

Figure 2

Cardiac hemodynamic function at 1, 3, 7, and 14 days after intraperitoneal DOM administration, compared with naïve control and 1- and 14-day saline-treated control rats: left ventricular developed pressure (LVDP) (A), dP/dt minimum (B), dP/dt maximum (C), and coronary flow at 10 mmHg diastolic pressure (D). Data are expressed as means ± SEM of 8 separate experiments. S, saline. *P < 0.05, **P < 0.01, and ***P < 0.001 versus naïve control; ^†P < 0.05, ^††P < 0.01, and ^†††P < 0.001 versus 1-day saline; ^‡P < 0.05, ^‡‡P < 0.01, and ^‡‡‡P < 0.001 versus 14-day saline.

Figure 3

Cardiac hemodynamic function at 7 and 14 days after intrahippocampal DOM administration, compared with naïve control and 14-day saline-treated control rats: LVDP (A), dP/dt minimum (B), dP/dt maximum (C), and coronary flow at 10 mmHg diastolic pressure (D). Data are expressed as means ± SEM of 8 separate experiments. S, saline. ***P < 0.001 versus naïve control; ^‡P < 0.05, ^‡‡P < 0.01, and ^‡‡‡P < 0.001 versus 14-day saline.

Figure 4

Isolated cardiac complex I (A), complex II-III (B), complex V (C), citrate synthase (D), and aconitase activities (E) at 1, 3, 7, and 14 days after intraperitoneal DOM administration (gray bars) in rats, compared with 1 and 14 days after saline administration in controls (black bars). Data are expressed as means ± SEM of 8 separate experiments. ^†P < 0.05, ^††P < 0.01, ^†††P < 0.001 versus 1-day saline; ^‡P < 0.05, ^‡‡P < 0.01, and ^‡‡‡P < 0.001 versus 14-day saline.

Figure 5

Isolated cardiac complex I (A), complex II-III (B), complex V (C), citrate synthase (D), and aconitase activities (E) at 7 and 14 days after intrahippocampal DOM administration (gray bars) in rats, compared with 14 days after saline administration in controls (black bars). Data are expressed as means ± SEM of 8 separate experiments. ^‡P < 0.05, ^‡‡P < 0.01, and ^‡‡‡P < 0.001 versus 14-day saline.

Figure 6

Histopathological changes in LV myocardium of saline- or DOM-treated rats, visualized using H&E stain. A: Normal histology of cardiomyocytes, 14 days after intraperitoneal saline. B: Equally intact tightly packed cardiomyocytes with cross-striations, 14 days after intrahippocampal saline. C: Reversible degenerative cardiomyopathy, evidenced by myocyte vacuolization (arrowheads indicate intracellular vacuoles displacing nuclei) and edema, 3 days after intraperitoneal DOM. D: Prominent hypercontraction bands associated with fiber derangement, 3 days after intraperitoneal DOM. E: Numerous interstitial inflammatory cells in proximity to necrotic cells typical of coagulation necrosis and evidence of interstitial edema, 7 days after intraperitoneal DOM. F: Phagocytosis of necrotic myocardium with inflammatory infiltrate and edema still present, at 14 days after intraperitoneal DOM. G: Hypercontraction bands with fiber derangement and edema, 7 days after intrahippocampal DOM. H: Muscle fibers disintegrated into eosinophilic granular material with extensive inflammatory infiltrate (arrowheads) and edema, 14 days after intrahippocampal DOM. Scale bars: 10 μm.

Figure 7

Both intraperitoneal and intrahippocampal delivery of DOM-induced cardiac fibrosis at 14 days after drug administration, as seen in representative photomicrographs of picro-Sirius-stained LV subendocardium sections. A: Saline control (intrahippocampal dosed) rats, with absence of Sirius red staining (absence of fibrosis). B and C: DOM intrahippocampal (B) and DOM intraperitoneal (C) dosed rats show evidence of interstitial fibrosis and reparative fibrosis. D–F: Evidence of increased LV perivascular fibrosis in DOM-dosed intrahippocampal (E) and intraperitoneal (F) animals, compared with saline-treated control (D) control showing modest collagen levels surrounding the coronary artery. Asterisk indicates reparative fibrosis. EL, endocardial lumen; IF, interstitial fibrosis; PF, perivascular fibrosis; VL vascular lumen. Scale bars: 10 μm.

Figure 8

Stimulatory effect of intraperitoneal (A, C, E) and intrahippocampal (B, D, F) DOM delivery in rats on circulating proinflammatory cytokine IL-1α, IL-1β, and TNF-α levels at 1- to 14-day termination points after administration of DOM (gray bars), compared with the respective saline-treated controls (black bars). Data are expressed as means ± SEM of 8 separate experiments. ^†P < 0.05, ^††P < 0.01 versus 1-day saline; ^‡P < 0.05, ^‡‡P < 0.01 versus 14-day saline.

Figure 9

Effect of intraperitoneal (A) and intrahippocampal (B) DOM delivery in rats on circulating GM-CSF levels at termination points 1, 3, 7, and 14 days after administration of DOM (gray bars), compared with saline-treated controls at 1 and 14 days (black bars). Data are expressed as means ± SEM of 8 separate experiments.