Morphological and biochemical characterization of basal and starvation-induced autophagy in isolated adult rat cardiomyocytes.

Abstract

Autophagy is simultaneously a mode of programmed cell death and an important physiological process for cell survival, but its pathophysiological significance in cardiac myocytes remains largely unknown. We induced autophagy in isolated adult rat ventricular cardiomyocytes (ARVCs) by incubating them in glucose-free, mannitol-supplemented medium for up to 4 days. Ultrastructurally, intracellular vacuoles containing degenerated subcellular organelles (e.g., mitochondria) were markedly apparent in the glucose-starved cells. Microtubule-associated protein-1 light chain 3 was significantly upregulated among the glucose-starved ARVCs than among the controls. After 4 days, glucose-starved ARVCs showed a significantly worse survival rate (19+/-5.2%) than the controls (55+/-8.3%, P<0.005). Most dead ARVCs in both groups showed features of necrosis, and the rate of apoptosis did not differ between the groups. Two inhibitors of autophagy, 3-methyladenine (3-MA) and leupeptin, significantly and dose-dependently reduced the viability of both control and glucose-starved ARVCs and caused specific morphological alterations; 3-MA reduced autophagic findings, whereas leupeptin greatly increased the numbers and the sizes of vacuoles that contained incompletely digested organelles. The knockdown of the autophagy-related genes with small interfering RNA also reduced the glucose-starved ARVCs viability, but rapamycin, an autophagy enhancer, improved it. Reductions in the ATP content of ARVCs caused by glucose depletion were exacerbated by the inhibitors while attenuated by rapamycin, suggesting that autophagy inhibition might accelerate energy depletion, leading to necrosis. Taken together, our findings suggest that autophagy in cardiomyocytes reflects a prosurvival, compensatory response to stress and that autophagic cardiomyocyte death represents an unsuccessful outcome due to necrosis.

• Authors: Rumi Maruyama, Kazuko Goto, Genzou Takemura, Koh Ono, Kazuya Nagao, Takahiro Horie, Akiko Tsujimoto, Hiromitsu Kanamori, Shusaku Miyata, Hiroaki Ushikoshi, Kenshi Nagashima, Shinya Minatoguchi, Takako Fujiwara, Hisayoshi Fujiwara
• Journal: American journal of physiology. Heart and circulatory physiology (Am J Physiol Heart Circ Physiol) Vol. 295 Issue 4 Pg. H1599-607 (Oct 2008) ISSN: 0363-6135 [Print] United States
• PMID: 18708438 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• LC3 protein, rat
• Leupeptins
• Microtubule-Associated Proteins
• RNA, Small Interfering
• 3-methyladenine
• Adenosine Triphosphate
• Glucose
• leupeptin
• Adenine
• Sirolimus

Topics

• Adenine (analogs & derivatives, pharmacology)
• Adenosine Triphosphate (metabolism)
• Animals
• Autophagy (drug effects, genetics)
• Cell Shape (drug effects)
• Cell Survival (drug effects)
• Cells, Cultured
• Dose-Response Relationship, Drug
• Glucose (deficiency)
• Leupeptins (pharmacology)
• Male
• Microtubule-Associated Proteins (metabolism)
• Myocytes, Cardiac (drug effects, metabolism, ultrastructure)
• Necrosis
• RNA Interference
• RNA, Small Interfering (metabolism)
• Rats
• Rats, Sprague-Dawley
• Sirolimus (pharmacology)
• Time Factors
• Vacuoles (ultrastructure)