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.

Fate of isolated adult cardiomyocytes undergoing starvation-induced autophagic degeneration

“…Uncontrolled increases in cytosolic Ca 2+ result in supercontraction of sarcomeres, which destroys the plasma membrane and cytoplasmic architecture-i.e., causes necrotic cell death. As shown in our study, 6 inhibitors of autophagy increased the incidence of cardiomyocyte necrosis accompanying ATP reduction. We therefore supposed that necrosis might be the result of energy depletion (primary necrosis) caused by suppression of the recycling needed for energy production via autophagy, though the possibility that ATP levels dropped as the result of cell rupture was not excluded.…”

“…In the heart, autophagic cardiomyocytes are observed under pathological conditions such as heart failure, pressure overload and myocardial ischemia and/or reperfusion, 2-5 but their functional significance remains controversial. In our recent study, 6 we examined the functional role of starvation-induced autophagy in isolated adult cardiomyocytes, a terminally differentiated cell with a highly specialized structure and function. We found that inhibiting the autophagic process by suppressing either phagophore formation using 3-methyladenine or the digestion step using leupeptin significantly reduced survival among cardiomyocytes; conversely, enhancing autophagy using rapamycin increased survival.…”

“…1). 6 After being cultured for 4 days with or without glucose, many cardiomyocytes showed features of necrosis-i.e., disrupted plasma membranes, swollen mitochondria containing dense bodies, and supercontracted sarcomeres. Autophagosomes were observed in both necrotic and living (normal in appearance) cardiomyocytes, and were far more numerous in glucose-starved cells.…”

FoxO Transcription Factors Promote Autophagy in Cardiomyocytes

“…Therefore, autophagy in cardiomyocytes is required for normal cellular function in the basal state. Inhibition of autophagy in cultured adult rat ventricular cardiomyocytes results in cell death, further supporting the essential role of autophagy in cardiomyocyte cell survival (17,18). Overall, autophagy is required for normal cellular function and for response to multiple types of stress in the neonatal and adult heart.…”

“…In the adult heart, autophagy is required for homeostasis, because cardiac specific loss of Atg5 leads to cardiac hypertrophy, left ventricular dilation, contractile dysfunction, and ultimately heart failure (16). During nutrient deprivation, autophagy is induced in the adult mouse heart in vivo and in cultured neonatal rat cardiomyocytes (13,17). Increased cardiac autophagy also occurs with dilated cardiomyopathy, heart failure, ischemia/reperfusion, aortic stenosis, and in hibernating myocardium (11, 18 -21).…”