Mitophagy‐regulated mitochondrial health strongly protects the heart against cardiac dysfunction after acute myocardial infarction

Abstract Autophagy including mitophagy serves as an important regulatory mechanism in the heart to maintain the cellular homeostasis and to protect against heart damages caused by myocardial infarction (MI). The current study aims to dissect roles of general autophagy and specific mitophagy in regulating cardiac function after MI. By using Beclin1+/−, Fundc1 knockout (KO) and Fundc1 transgenic (TG) mouse models, combined with starvation and MI models, we found that Fundc1 KO caused more severe mitochondrial and cardiac dysfunction damages than Beclin1+/− after MI. Interestingly, Beclin1+/− caused notable decrease of total autophagy without detectable change to mitophagy, and Fundc1 KO markedly suppressed mitophagy but did not change the total autophagy activity. In contrast, starvation increased total autophagy without changing mitophagy while Fundc1 TG elevated total autophagy and mitophagy in mouse hearts. As a result, Fundc1 TG provided much stronger protective effects than starvation after MI. Moreover, Beclin1+/−/Fundc1 TG showed increased total autophagy and mitophagy to a level comparable to Fundc1 TG per se, and completely reversed Beclin1+/−‐caused aggravation of mitochondrial and cardiac injury after MI. Our results reveal that mitophagy but not general autophagy contributes predominantly to the cardiac protective effect through regulating mitochondrial function.

various pathological conditions such as hypertrophy, heart failure and ischaemic cardiomyopathy. 3,4 Growing evidence suggests that autophagy is generally a protective response in ischaemic heart diseases especially in acute myocardial infarction (MI). 5,6 For instance, starvation-induced autophagy preserved ATP level in MI mouse hearts and reduced infarct size, while inhibition of autophagy abolished starvation-caused cardiac protection. 7 Beclin1 is a key component of mammalian autophagy regulators and is widely expressed in tissues including the heart. [8][9][10] The heterozygous Beclin1 knockout mice had reduced autophagy and displayed further heart damage after MI. 10 By and large, appropriately elevated autophagy may promote the survival of cardiomyocytes and protect the heart against cardiac dysfunction during cardiac stresses, and inhibition or deficiency of autophagy aggravates the cardiac dysfunction. 4,11,12 Autophagy is originally believed to be a non-selective process, but later studies have identified specifically selective targets of autophagy such as peroxisomes, endoplasmic reticulum and mitochondria. [13][14][15] Among them, selective removal of mitochondria is termed mitophagy. 16,17 Impaired mitochondria may produce excessive reactive oxygen species (ROS) and release cell death-inducing factors therefore will be removed by autophagy/mitophagy to prevent further damage occurred to mitochondria or to the cell. 17,18 Mitophagy is regulated through PINK1/Parkin pathway or mitophagy receptors.

Several mitophagy receptors have been identified in mammalian
cells including Bnip3L/Nix, Bnip3 and Fundc1. [19][20][21] Similar with general autophagy, aberrant mitophagy is associated with a wide variety of cardiovascular pathologies such as hypertrophy, heart failure and MI. 22,23 At the early stage of MI, mitophagy was activated through PINK1/Parkin pathway, and Parkin deficient mice exhibited decreased mitophagy, accumulation of dysfunctional mitochondria and reduced survival after MI. 24 Likewise, cardiac knockout of Fundc1, the specific hypoxia-related mitophagy receptor, aggravates cardiac dysfunction after MI. 25 These studies suggest that selective mitophagy also plays a critical role in cardiac diseases.
During myocardial infarction, the coronary artery is sharply interrupted, resulting in severe ischaemia of the corresponding myocardium and eventually leading to cardiac damage and dysfunction.
Autophagy is largely initiated within 1 week after MI (acute MI) and returned to normal level 3 weeks after MI. 26 A growing body of evidence reveals that suppression of autophagy causes exacerbation of the myocardial damage by acute MI. 10,27 The heart is an organ with high bioenergetics demands which are mainly supplied by mitochondria, [28][29][30][31][32] so it requires fine-regulated autophagy/mitophagy to maintain healthy mitochondrial population and the cardiac function after MI. 6,7 Emerging evidence indicates that autophagy/mitophagy is activated and either the increased autophagy or mitophagy protects the myocardium. 6,24 However, there is no evidence to show whether the general autophagy and selective mitophagy equally protect the heart or are replaceable to each other in cardiac protective effect after acute MI. We believe that accurately evaluating the contribution of autophagy and mitophagy to their cardiac protective function after acute MI is of great importance for the modulation of autophagy/mitophagy as a potential therapeutic target for ischaemic heart diseases. Therefore, the present study employs Beclin1 +/− mice, Fundc1 knockout mice and Fundc1 transgenic mice, combined with starvation and acute MI mouse models, to dissect the role of general autophagy and selective mitophagy in cardiac function and after acute MI, thus to expand our understanding of autophagy/mitophagy and to provide potential therapeutic strategies for the treatment of acute MI and ischaemia-related cardiac diseases.

| MATERIAL S AND ME THODS
All supporting data are available within the article. Methods are provided as online data supplement.

| Both autophagy and mitophagy are increased in mouse hearts after myocardial infarction
To understand the role of general autophagy and selective mitophagy in regulating the heart function after acute myocardial infarction (MI), we established mouse MI model by ligating the left main descending coronary artery. Echocardiography showed a decline in left ventricular systolic function in mice at 1 and 7 days after MI compared with Sham mice, as evidenced by decreased ejection fraction (EF) and fractional shortening (FS) ( Figure S1A and Table S1).
Moreover, cardiac remodelling was evidenced by picric acid-sirius red staining of interstitial collagen deposition at 7 days after MI ( Figure S1B). In hearts at 1, 3, 5 and 7 days after MI, protein levels of autophagy markers LC3 II and Beclin1 were significantly increased as compared with sham control mouse hearts ( Figure 1A,B), indicating the increased activity of the general autophagy after acute MI.
However, protein levels of mitophagy receptor Fundc1, mitochondrial inner membrane protein Tim23 and mitochondrial outer membrane protein Tom20 were all significantly decreased ( Figure 1A,B).
Given the increased autophagic markers and widely decreased mitochondrial proteins, the reduction of Fundc1 protein may be caused by the decreased amount of mitochondria by increased autophagy.
We then further measured mitochondrial protein levels from isolated mitochondria in mouse hearts after acute MI. Comparing with mitochondria from sham control mouse hearts, both LC3 II and Fundc1 proteins in mitochondria from MI hearts were significantly increased as normalized by Tim23 protein ( Figure 1C,D), indicating the increased activity of mitophagy after acute MI.
To further validate the change of mitophagy in MI hearts, we established the MI model with mito-Keima mouse, a mouse model with mitochondria-targeted expression of the pH-indicator protein Keima. 33,34 Compared with mitochondria in sham control mouse hearts, increased amount of lysosome-engulfed mitochondria was observed in MI hearts at 1, 3, 5 and 7 days, as indicated by the in-   Figure 2A and Table S2). One day after MI, both Beclin1 +/− mice and Fundc1 KO mice showed lower EF and FS than wild-type MI mice, and Fundc1 KO mice showed the lowest EF and FS ( Figure 2A and Table S2). Consistently, after acute MI, while both Beclin1 +/− and confirming that Fundc1 deficiency causes more severe cardiac damage than Beclin1 deficiency after acute MI. Then, a question arises of whether the autophagy and mitophagy are comparably damaged or kept in the two different mouse models, Beclin1 +/− and Fundc1 KO mouse models.
To dissect the general autophagy and mitophagy levels in hearts, we first measured LC3 II and mitochondrial protein levels.
At basal condition, heterozygous deletion of Beclin1 suppressed the total LC3 II protein level, indicating the inhibited autophagy in Beclin1 +/− mouse hearts ( Figure 2D). Similarly, although total LC3 II level was increased in Beclin1 +/− mouse hearts after acute MI, it was lower than the increased LC3 II level in wild-type mouse hearts after acute MI ( Figure 2D). However, total LC3 II protein level in Fundc1 KO mouse hearts showed no difference compared with wild-type mouse hearts at either basal condition or after acute MI ( Figure 2D). These data suggest that the general autophagy is inhibited in Beclin1 +/− mouse hearts but keeps intact, at least largely if not totally, in Fundc1 KO mouse hearts. In contrast, mitochondrial proteins such as Tom20 and Fundc1 in Beclin1 +/− mouse hearts showed no difference with those in wild-type hearts at basal condition and decreased similarly both in Beclin1 +/− and wild-type mouse hearts after acute MI ( Figure 2D), indicating that mitophagy activity is not significantly changed in Beclin1 +/− mouse hearts. Unsurprisingly, in Fundc1 KO mouse hearts, Tom20 largely increased as compared with wild-type mouse hearts ( Figure 2D). Although Tom20 protein level was decreased after acute MI in the hearts of all three mouse models, it was the highest in Fundc1 KO mouse hearts ( Figure 2D), indicating that mitophagy activity is largely inhibited in Fundc1 KO mouse hearts while keeps largely intact in Beclin1 +/− mouse hearts. Mitochondrial LC3 II protein level showed no difference between Beclin1 +/− and wild-type mice, but was largely suppressed in Fundc1 KO mice compared with wild-type mice at both sham level and after acute MI ( Figure 2E), suggesting that Fundc1 KO suppresses mitophagy while Beclin1 +/− hardly contributes to mitophagy. These results suggest that Beclin1 deficiency mainly inhibits the general autophagy but scarcely changing mitophagy, and Fundc1 deficiency reduces mitophagy with nearly unaltered total autophagy.
Previous studies showed that in addition to regulating mitophagy, Fundc1 has multiply cellular functions such as mediating mitochondrial fission, mitochondria-associated endoplasmic reticulum membranes and mitochondrial metabolism. [35][36][37] To further clarify that Fundc1 deficiency aggravates cardiac function through inhibiting mitophagy, we employed a synthetic cell-penetrating peptide containing the unphosphorylated Tyr18 at LIR motif of Fundc1, which inhibits the Fundc1/LC3 interaction thus specifically blocking  Figure 3C and Table S3). Also, peptide P significantly increased serum LDH level and cardiac infarct size after acute MI comparing to peptide C ( Figure 3D,E). Collectively, these data indicate that mitophagy plays more important role in protecting cardiac function after acute MI than general autophagy.

| Fundc1 deficiency aggravates mitochondrial dysfunction after acute MI
To know cardiac mitochondria function in Beclin1 +/− and

| Upregulation of mitophagy/autophagy by Fundc1 overexpression provides stronger protective effect on the heart after myocardial infarction than upregulation of autophagy by starvation
Next, we seek to know if increasing autophagy/mitophagy ameliorates cardiac injury after acute MI, and to further dissect the possible contribution by autophagy/mitophagy. Wild-type mice fasted for 1 day were used to induce general autophagy and the acute MI also stimulated cardiac mitophagy activity ( Figure 5B).

Importantly, mitochondrial LC3 II in Fundc1 TG mouse hearts
increased dramatically as compared with that in wild-type and starvation mouse hearts after acute MI ( Figure 5B). Together, our results suggest that, although starvation and Fundc1 TG induce comparably higher general autophagy than wild type in the hearts after acute MI, mitophagy was not significantly changed in starvation-treated mouse hearts compared with wild-type mice, that is only Fundc1 TG increases specific mitophagy, no matter under basal condition or after acute MI. Echocardiography showed that Beclin1 +/− /Fundc1 TG mice largely preserved cardiac function from acute MI compared with Beclin1 +/− mice, indicated by the increased EF and FS ( Figure 6C).

And after acute MI, the EF, FS and LVIDs levels were similar in
Beclin1 +/− /Fundc1 TG mice as in Fundc1 TG mice ( Figure 6C and Table S5). Moreover, Fundc1 TG completely suppressed Beclin1 +/−caused increase of cardiac infarct size and the serum LDH level after acute MI ( Figure 6D,E), illustrating that Fundc1 overexpression totally reverses Beclin1 deficiency caused exacerbation of cardiac injury after acute MI. At mitochondrial level, the mitochondrial respiratory function, Δψm and mitochondrial ROS production were also completely preserved in Beclin1 +/− /Fundc1 TG mouse hearts after acute MI, compared with that in Beclin1 +/− mice, to levels similar as in Fundc1 TG mice ( Figure 6F-H). Together, our results suggest that both general autophagy and mitophagy protect the heart against MI-caused cardiac injury, however, mitophagy per se at least predominantly, if not totally, contributes to this cardiac protective effect presumably through regulating mitochondrial function.

| DISCUSS ION
The present study found that general autophagy and specific mi- In the heart, normal autophagy activity is vital for the maintenance of cardiac function at both physiological and pathological conditions. 6,11,[38][39][40] It has been reported that autophagy activity was largely activated in ischaemic cardiac diseases especially after acute MI in patients and in animal models. 41,42 Here, our present work found that general autophagy was increased from 1 day to 7 days after acute MI, and starvation stimulated autophagy activ-   (Figure 6), suggesting that preserving mitophagy per se totally compensates autophagy deficiency caused mitochondrial and cardiac dysfunction. Together, the present study found that the mitophagy contributes predominantly, if not totally, to the protective effect of the general autophagy on cardiac function after acute MI.
One of the limitations of our present study is the animal models we used. We use Beclin1 +/− mice to show the inhibition of autophagy while this animal model could also regulate mitophagy. Likewise, Fundc1 KO mouse is used as the mitophagy-deficient mouse model while there are other mitophagy regulators in cardiomyocytes. To validate the models, we measure changes of both general autophagy and mitophagy in all animal models after acute MI. We found that the general autophagy is inhibited but mitophagy activity is not significantly changed in Beclin1 +/− mouse hearts comparing with wild-type mice after acute MI. And in contrast, mitophagy activity is inhibited but the general autophagy is kept at least largely intact in Fundc1 KO mouse hearts comparing with wild-type mice. However, further studies and experimental models are needed to more precisely dissect the intertwined roles of general autophagy and specific mitophagy in regulating cardiac functions.
In summary, we demonstrate that general autophagy and specific mitophagy are upregulated after acute MI. While the upregulation of either general autophagy or mitophagy protects the heart from MI-caused damage, mitophagy contributes predominantly to the protective effect on mitochondrial and cardiac function. Our results provide new insights into the functional roles of general autophagy and specific mitophagy with the predominant contribution of mitophagy in the heart, and further suggest the more promising therapeutic strategy by regulating the mitophagy activity but not general autophagy activity during MI or ischaemic heart diseases.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request