title: 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: 1Division of Cardiology, Gifu University Graduate School of Medicine, Gifu, Japan.
authors: Rumi
authors: R
authors: Maruyama
authors: Kazuko
authors: K
authors: Goto
authors: Genzou
authors: G
authors: Takemura
authors: Koh
authors: K
authors: Ono
authors: Kazuya
authors: K
authors: Nagao
authors: Takahiro
authors: T
authors: Horie
authors: Akiko
authors: A
authors: Tsujimoto
authors: Hiromitsu
authors: H
authors: Kanamori
authors: Shusaku
authors: S
authors: Miyata
authors: Hiroaki
authors: H
authors: Ushikoshi
authors: Kenshi
authors: K
authors: Nagashima
authors: Shinya
authors: S
authors: Minatoguchi
authors: Takako
authors: T
authors: Fujiwara
authors: Hisayoshi
authors: H
authors: Fujiwara
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001343:Autophagy / Q000187:drug effects / Q000235:genetics; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005947:Glucose / Q000172:deficiency*; D007976:Leupeptins / Q000494:pharmacology; D008297:Male; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D009336:Necrosis; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020123:Sirolimus / Q000494:pharmacology; D013997:Time Factors; D014617:Vacuoles / Q000648:ultrastructure*
chemical_list: D000225:Adenine; D000255:Adenosine Triphosphate; D005947:Glucose; D007976:Leupeptins; D008869:Microtubule-Associated Proteins; D034741:RNA, Small Interfering; D020123:Sirolimus; C526109:Anxa3 protein, rat; C025946:3-methyladenine; C032854:leupeptin