Original articleStimulation of mitochondrial biogenesis and autophagy by lipopolysaccharide in the neonatal rat cardiomyocyte protects against programmed cell death
Introduction
Lipopolysaccharide (LPS, endotoxin) is a major component of bacterial outer walls that can cause profound and diverse effects in mammalian cells. It is the mediator of host responses that can lead to sepsis and ultimately multi-organ dysfunction. Cardiac myocyte programmed cell death (apoptosis) and depression of cardiac function are recognized sequelae of LPS-induced toxemia. In whole animals, cardiac dysfunction induced by LPS exposure is postulated to be a direct result of the activation of myocyte caspases and apoptosis [1]. Indeed, sub-lethal doses of LPS may condition and confer resistance to subsequent ischemia or endotoxemic injury by reprogramming myocardial gene expression [2].
Other investigators have reported an increase in TNF-α production and apoptosis when adult rat cardiomycytes are exposed to LPS [3], [4]. Our laboratory and others have demonstrated the response of the neonatal rat cardiomyocyte to LPS is more robust [4], [5]. Although the neonatal cells also produce TNF-α, levels of LPS, far exceeding those necessary to induce TNF-α, release, do not induce apoptosis. These cells are fully capable of instigating apoptosis in response to multiple other stimuli [6], [7], [8].
In vivo administration of LPS rapidly induces oxidative stress in the rat heart [9]. The mitochondrion is the principal site of generation of reactive species and the mitochondrial genome is particularly susceptible to oxidative damage. In our neonatal rat cardiomyocyte system, we demonstrate that LPS treatment leads to a loss of mitochondrial membrane potential (Δψ) which is temporally associated with an increase in the level of uncoupling protein 3 (UCP3). However, these cells remain viable with no measurable increase in cell death.
We also demonstrate that, as in the in vivo model, many markers of mitochondrial biogenesis are activated [9]. Mitochondrial biogenesis requires the coordinated expression of both nuclear and mitochondrially encoded genes. LPS treatment stimulates an increase in transcription of mitochondrial transcription factor A (Tfam), increase in nuclear accumulation of redox-sensitive nuclear respiratory factor 1 (NRF-1), and increase in expression of peroxisome proliferator-activated receptor γ co-activator 1 (PGC-1).
While mitochondrial biogenesis is stimulated in this model, we also observed that LPS increased intracellular degradation of cytoplasmic components, including damaged mitochondria, a process known as autophagy, or macroautophagy. Autophagy was assessed by monitoring the levels of a mammalian protein specifically associated with autophagosomes, LC3-II [10]. Microtubule-associated light chain 3 (LC3), a homologue of the yeast protein Atg8, which is cleaved generating an 18-kDa soluble form of the protein, LC3-I. LC3-I is further modified to a membrane-bound, 16-kDa form, LC3-II. Furthermore, pharmacological inhibition of autophagy with 3-methyladenine (3-MA) in the presence of LPS stimulates apoptosis, as assessed by an increase in caspase-3 like activity, but has no effect upon the loss of mitochondrial membrane potential.
Section snippets
Primary cell culture
Neonatal rat ventricular cardiomyocytes were prepared according to McMillin et al. [11] using 1- to 2-day-old Sprague-Dawley pups. Cells were plated at a density of 2 × 106 cells/60-mm dish and maintained for 48 h in DMEM containing 0.3 g/l glutamine, 4.5 g/l glucose and 10% calf serum before LPS exposure. LPS purified from Escherichia coli strain K-245 (Sigma) with a serotype of K1 O7 [12] was administered to cells in culture (1 μg/ml) in serum-containing media. Inhibition of autophagy with 3-MA
Oxidative stress, mitochondrial membrane potential, and UCP3
DCFH-DA enters cells and produces a fluorescent signal after intracellular oxidation by ROS such as hydrogen peroxide and hydroxyl radical [18]. DHE also freely enters cells and is oxidized by ROS, particularly superoxide, to yield fluorescent ethidium that can pass into the nucleus and subsequent binding to the DNA results in an amplified fluorescent signal [18]. Our results demonstrate that the neonatal rat cardiomycytes generated ROS in response to LPS exposure (Fig. 1).
LPS exposure also
Discussion
A significant body of evidence indicates that ROS production is increased during sepsis and these reactive species are involved in cellular damage. Increased mitochondrial ROS formation has been demonstrated to contribute to increased ROS levels in the heart and a temporal relationship demonstrated between this ROS production and tissue damage in LPS-treated animals [9], [20]. Our previous studies in neonatal rat cardiomyocytes demonstrated a rapid (within 1 h) increase in the expression of the
Acknowledgments
This work was supported by the U.S. Army (grant nos. DAMD17-01-2-0047 and DAMD W81XWH-04-02-0035).
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