Time‐dependent differential effects of fasting on cardiac autophagy and mitophagy

Objectives Alternate‐day fasting or starvation appears beneficial to the heart, but the underlying mechanism remains speculative. Starvation activates general autophagy, which may contribute to the cardioprotective effect of fasting. However, it is not clear whether mitochondrial degradation or mitophagy is involved in this process. Cellular proteome studies have shown that cytosolic proteins are degraded early during starvation and mitochondria are degraded much later. An increase in mitophagy in certain conditions has demonstrated cardio‐protective and anti‐aging effects. However, degradation of too many mitochondria can also compromise cell survival and cardiac function, highlighting the importance of a tightly controlled mitophagy response to preserve cardiac function in stressful conditions. The goal of this study is to determine the functional state of mitophagy in the heart during a 24‐hour or 48‐hour starvation. Methods To demonstrate the occurrence of mitophagy in the heart, we used a novel mitophagy reporter transgenic mouse line expressing mito‐Rosella, a dual‐emission biosensor comprising a pH‐stable red fluorescent protein (RFP) linked to a pH‐sensitive green fluorescent protein (GFP). These mice were subjected to either 24‐hours or 48‐hours of fasting. To assess mitophagy flux, these animals were also treated with pepstatin A/E64d, membrane‐permeable cathepsin inhibitors that lead to an accumulation of autolysosomes. Following treatment, animals were sacrificed, and cardiac tissues were harvested for confocal microscopic analysis of mitophagy events, mitochondrial isolation, and western blot analysis of LC3‐II protein levels. Mitophagy events were seen as red puncta on the overlaid confocal images. LC3‐II is a marker for autophagosome and autolysosomes, the vacuoles that sequester cellular components for degradation. Results A 24‐hour fasting increased mitophagy flux as demonstrated by a 44.2% increase in the number of mitophagy events in heart sections. The average size of mitophagy events within studied myocytes increased by 13.4%. Western blot analysis showed an increase in LC3‐II protein levels in both total cardiac tissue lysates and the mitochondrial fractions, suggesting that autophagy and mitophagy were enhanced in parallel. However, after 48 hours of starvation, mitophagy events decreased by 50.1% compared to events at 24 hours of fasting and decreased by 28% compared to the control fed animals. The average area of each mitophagy event after 48 hours of fasting decreased by 39.9% compared to that at 24 hours and decreased by 31.9% compared to control. Interestingly, Western Blot analysis showed that LC3‐II protein levels were increased in the total cardiac tissue lysates but reduced in the mitochondrial fractions, suggesting that the 48‐hour fasting enhanced general autophagy but inhibited mitophagy. Conclusion These results demonstrate that fasting‐caused energy deficiency accelerates mitophagy flux in the heart along with an increase in total autophagy during the first 24 hours. However, sustained nutrient deprivation for 48 hours significantly decreases mitophagy flux although autophagy continues at a high rate, suggesting that mitophagy must be mechanistically separable from general autophagy and be regulated by different signaling pathways. Support or Funding Information National Institute of Health