Stimulation of mitochondrial biogenesis and autophagy by lipopolysaccharide in the neonatal rat cardiomyocyte protects against programmed cell death

doi:10.1016/j.yjmcc.2007.10.013

Journal of Molecular and Cellular Cardiology

Volume 44, Issue 2, February 2008, Pages 411-418

https://doi.org/10.1016/j.yjmcc.2007.10.013 Get rights and content

Abstract

Adult rat cardiomyocytes in culture respond to sub-lethal doses of lipopolysaccharides (LPS) by activation of pathways including the production of TNF-α and increased apoptosis. We and others have demonstrated a protective phenotype for neonatal rat cardiomyocytes to LPS. Concentrations of LPS far exceeding those necessary to induce TNF-α release do not induce apoptosis in the neonatal cells, although these cells are fully capable or inducing apoptosis in response to multiple other stimuli. In neonatal cells, 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). Cells remain viable with no measurable increase in apoptotic or necrotic cell death. Many markers of mitochondrial biogenesis are also activated. LPS treatment stimulates an increase in the (i) transcription of mitochondrial transcription factor A (Tfam), (ii) nuclear accumulation of redox-sensitive nuclear respiratory factor 1 (NRF-1), and (iii) expression of peroxisome proliferator-activated receptor γ co-activator 1 (PGC-1). We also observed that LPS increased intracellular autophagy. Autophagy was assessed by monitoring the levels of a mammalian protein specifically associated with autophagosomes, microtubule-associated light chain 3 (LC3). Furthermore, inhibition of autophagy in the presence of LPS stimulates markers of apoptosis. Our data suggest that the protective response of neonatal cells to LPS is multi-faceted at the level of the mitochondrion. Viable cells replace dysfunctional mitochondria by mitochondrial biogenesis and the extent of the damage limited by the rapid removal of damaged organelles by the stimulation of autophagy.

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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Original articleStimulation of mitochondrial biogenesis and autophagy by lipopolysaccharide in the neonatal rat cardiomyocyte protects against programmed cell death

Abstract

Introduction

Section snippets

Primary cell culture

Oxidative stress, mitochondrial membrane potential, and UCP3

Discussion

Acknowledgments

J Mol Cell Cardiol

Methods Toxicol

Methods Enzymol

Biophys J

J Mol Cell Cardiol

FEBS Lett

FEBS Lett

J Biol Chem

Int J Biochem Cell Biol

Mutat Res

Biochem Mol Med

Brain Res Mol Brain Res

FEBS Lett

Cell

Biochim Biophys Acta

Comp Biochem Physiol A Mol Integr Physiol

Mol Aspects Med

Int J Biochem Cell Biol

Ventricular myocyte caspases are directly responsible for endotoxin-induced cardiac dysfunction

Circulation

Original article
Stimulation of mitochondrial biogenesis and autophagy by lipopolysaccharide in the neonatal rat cardiomyocyte protects against programmed cell death