Ischemic stroke brain sends indirect cell death signals to the heart

Abstract

Background and purpose: Ischemic stroke is a leading cause of mortality and morbidity in the world and may be associated with cardiac myocyte vulnerability. However, it remains uncertain how an ischemic brain contributes to cardiac alternations. Here, we used experimental stroke models to reveal the pathological effects of the ischemic brain on the heart.

Methods: For the in vitro study, primary rat neuronal cells were subjected to 90-minute oxygen-glucose deprivation (OGD). Two hours after OGD, the supernatant was collected and cryopreserved until further biological assays. Primary rat cardiac myocytes were exposed to ischemic-reperfusion injury and subsequently to the supernatant derived from either the OGD or non-OGD-exposed primary rat neuronal cells for 2, 6, 24, or 48 hours. Thereafter, we measured cell viability and mitochondrial activity in rat cardiac myocytes. For the in vivo study, we subjected adult rats to transient middle cerebral artery occlusion, and their brains and hearts were harvested for immunohistochemical analyses at 3 months later.

Results: The supernatant from the OGD, but not the non-OGD-exposed primary rat neuronal cells, caused significant reduction in cell viability and mitochondrial activity in rat cardiac myocytes. Ischemic stroke animals displayed phenotypic expression of necrosis, apoptosis, and autophagy in their hearts, which paralleled the detection of these same cell death markers in their brains.

Conclusions: Ischemic stroke was accompanied by cardiac myocyte death, indicating a close pathological link between brain and heart. These results suggest a vigilant assessment of the heart condition in stroke patients, likely requiring the need to treat systemic cardiac symptoms after an ischemic brain episode.

Keywords: apoptosis; autophagy; brain ischemia; myocytes, cardiac; necrosis.

Figure 1

Immunocytochemical analyses of PRNCs after OGD condition. Cell death markers of necrosis, apoptosis, autophagy were elevated after OGD as evidenced by significantly increments in TNF alpha, Caspase 3, Fas Ligand, and MAP1L3CA labeled cells compared to normal condition. Scale bar represents 50 μm.

Figure 2

Immunocytochemical analyses of RCMs after I/R injury and treatment with supernatant harvested from PRNCs exposed to OGD. Cell death markers, including TNF alpha, Caspase 3, Fas Ligand, and MAP1LC3A, were elevated after I/R injury and treatment with supernatant harvested from PRNCs exposed to OGD compared to the addition of supernatant from non-OGD-exposed PRNCs. Scale bar represents 50 μm.

Figure 3

Cell viability and mitochondrial activity in RCMs after I/R injury and supernatant from OGD-exposed PRNCs. Cell viability of RCMs as revealed by calcein assay was down-regulated at 24 hours after I/R injury and treatment with supernatant harvested from PRNCs exposed to OGD compared to the addition of supernatant from non-OGD-exposed PRNCs. Similarly, mitochondrial activity using the MTT assay revealed that the relative mitochondrial reductase activity of RCMs was significantly reduced at 48 hours after I/R injury and treatment with supernatant harvested from PRNCs exposed to OGD compared to the addition of supernatant from non-OGD-exposed PRNCs. Interestingly, both calcein and MTT assays revealed that I/R injury alone without the supernatant from the OGD-exposed PRNCs (DMEM treatment group represents the mean values over 48 hours) did not cause cardiac myocyte death.

Figure 4

Immunohistochemical analyses of brain and heart in ischemic stroke rats. In the brain, necrotic, apoptotic, and autophagic cells were mostly detected in the the ischemic stroke ipsilateral hemisphere. Whereas Caspase 3 and MAP1LC3A were observed in both hemispheres, the contralateral side immunoreactivity was less intense than ipsilateral side. The heart from the ischemic stroke animal also displayed all cell death markers. The immunoreactivity in sham animals was less intense than in stroke animals. Scale bar represents 50 μm.