Microtubule alteration is an early cellular reaction to the metabolic challenge in ischemic cardiomyocytes

Abstract

Cytoskeleton damage, particularly microtubule (MT) alterations, may play an important role in the pathogenesis of ischemia-induced myocardial injury. However, this disorganization has been scarcely confirmed in the cellular context. We evaluated MT network disassembly in myoblast cell line H9c2 and in neonatal rat cardiomyocytes in an in vitro substrate-free hypoxia model of simulated ischemia (SI). After different duration of SI from 30 up to 180 min, the cells were fixed and the microtubule network was revealed by immunocytochemistry. The microtubule alterations were quantified using a house-developed image analysis program. Additionally, the tubulin fraction were extracted and quantified by Western blotting. The cell respiration, the release of cellular LDH and the cell viability were evaluated at the same periods. An early MT disassembly was observed after 60 min of SI. The decrease in MT fluorescence intensity at 60 and 90 min was correlated with a microtubule disassembly. Conversely, SI-induced significant LDH release (35%) and decrease in cell viability (34%) occurred after 120 min only. These results suggest that the simulated ischemia-induced changes in MT network should not be considered as an ultrastructural hallmark of the cell injury and could rather be an early ultrastructural correlate of the cellular reaction to the metabolic challenge.

Limited forward trafficking of connexin 43 reduces cell-cell coupling in stressed human and mouse myocardium

“…Oxidative stress is known to interfere with microtubule integrity and growth (33)(34)(35). Using HeLa cells, we performed confocal immunofluorescent detection of α-tubulin and EB1 ( Figure 3A).…”

“…There have been several reports of deleterious effects on the cardiomyocyte cytoskeleton, particularly in microtubules, as a result of ischemic and oxidative stress (33)(34)(35). Given the relatively short half-life of Cx43 hemichannels at the plasma membrane (36)(37)(38), their dependence on microtubules for delivery to the plasma membrane (18,19,24), and the diminished gap junction coupling associated with cardiac pathologies (3)(4)(5), we investigated whether forward trafficking of Cx43 was perturbed in the stressed/diseased myocardium, thus contributing to losses in cell-cell electrical coupling.…”

Phosphorylation of DYNLT1 at Serine 82 Regulates Microtubule Stability and Mitochondrial Permeabilization in Hypoxia

“…Immunocytochemical staining was performed as described previously (Feng et al, 2007;Vandroux et al, 2004). Cells were cultured on coverslips (10-mm diameter) and rinsed twice in prewarmed (37°C) PBS.…”

“…Recent studies have showed that in case of ischemia-reperfusion or oxygen-glucose deprivation, the MTs are more prone to hypoxic damage than the actin filaments (Sato et al, 1993). In addition, it is known that early disruption of the cytoskeleton ultrastructure correlates with the cellular reaction to a metabolic challenge (Hein et al, 1995;Vandroux et al, 2004), and thus promoting irreversible cell damage. It was reported that the homeostasis of MT forms relies on MT-associated proteins (MAPs) (Cassimeris, 1999).…”

The p38/MAPK pathway regulates microtubule polymerization through phosphorylation of MAP4 and Op18 in hypoxic cells

“…Cytoskeletal damage, including MT alterations, has been linked with various pathological conditions. Disruption of the MT network has been reported in ischemia as an early ultrastructural change correlated with the cellular reaction to a metabolic challenge [11][12][13], and irreversible cell damage may be associated with the collapse of the MT cytoskeleton [14]. Proliferation of the MTs with taxol has been reported to protect against hypoxia/re-oxygenation injury [15], whereas the MT disruptor, colchicine, has been shown to abolish the protective effect of ischemic pre-conditioning [16,17].…”