MCL1 is critical for mitochondrial function and autophagy in the heart

Abstract

MCL1 (myeloid cell leukemia sequence 1 [BCL2-related]) is an anti-apoptotic BCL2 family protein that is upregulated in several human cancers. In malignancies, overexpression of MCL1 promotes cell survival and confers chemotherapeutic resistance. MCL1 is also highly expressed in normal myocardium, but the functional importance of MCL1 in myocytes has not been explored. We recently discovered that MCL1 plays an essential role in myocardial homeostasis and autophagy. Here, we discuss how loss of MCL1 in the adult mouse heart leads to mitochondrial dysfunction, impaired autophagy and development of heart failure.

Although MCL1 exhibits functional and structural similarities with anti-apoptotic BCL2 and BCL2L1/BCL-X_L, its short half-life and lack of a BH4 domain make it unique among the BCL2 proteins. Moreover, BCL2, BCL2L1 and MCL1 are often co-expressed in the same tissue, but knockout studies have revealed that their physiological roles are different. To investigate the functional role of MCL1 in the heart, we generated inducible, cardiac-specific Mcl1 knockout mice. We discovered that loss of MCL1 in the adult heart leads to rapid heart failure and early mortality in the absence of other stressors. Early contractile dysfunction culminates in cardiac hypertrophy, fibrosis and inflammation. Surprisingly, although MCL1 is a well-known anti-apoptotic protein, we found that loss of MCL1 did not significantly increase caspase-activation, PARP cleavage, or AIFM/AIF translocation. Instead, ultrastructural analysis of cardiac sections from MCL1-deficient hearts revealed the presence of swollen mitochondria and ruptured myocytes consistent with mitochondrial permeability transition pore opening and necrotic cell death. These findings suggest that loss of MCL1 leads to necrotic rather than apoptotic cell death. Additional studies revealed that MCL1 is not a direct regulator of mitochondrial permeability transition pore opening and necrotic cell death. Rather, they are activated as a consequence of accumulating dysfunctional mitochondria.

The presence of damaged mitochondria in the cell is normally a strong signal to activate autophagy since such mitochondria can release prodeath proteins and produce excess reactive oxygen species. Removal of these aberrant mitochondria by autophagosomes can enhance cell survival by preventing activation of cellular death pathways. Cardiac myocytes, which contain extensive mitochondrial networks, routinely clear damaged mitochondria to maintain ATP production and prevent excessive reactive oxygen species output. Surprisingly, despite evidence of extensive mitochondrial damage after loss of MCL1, these cells do not activate autophagy. Instead, experiments assessing baseline and autophagic flux demonstrate that activation of autophagy is impaired in MCL1-deficient hearts. In addition, we found that MCL1-deficient hearts are incapable of activating autophagy in response to stressors such as exercise. This defect in autophagy is observed well before we see severe mitochondrial dysfunction and heart failure. Therefore, it is unlikely that the impairment in autophagy is a consequence of the mitochondrial dysfunction and cellular damage that occur upon loss of MCL1. Interestingly, MCL1 is not a direct activator of autophagy. Overexpression of MCL1 does not increase baseline or glucose starvation-induced autophagy in myocytes. At present, it is unclear how MCL1 regulates autophagy in the adult myocardium.

The selective clearance of dysfunctional mitochondria is regulated by the PINK1-PARK2/Parkin pathway. When mitochondria become damaged and lose their membrane potential, PINK1 accumulates on the mitochondria. This leads to recruitment of the E3 ubiquitin ligase PARK2, which then marks these mitochondria for degradation by ubiquitinating proteins on the mitochondrial outer membrane. We found that MCL1-deficiency in the heart is associated with a decrease in mitochondrial PINK1 levels. As a result, PARK2 is not recruited to mitochondria in MCL1-deficient hearts and accumulates in the cytosol. Clearly, these findings suggest that the presence of MCL1 is critical for functional PINK1-PARK2-mediated mitophagy.

MCL1 exists in both the outer mitochondrial membrane and in the matrix, where they exert different functions. The outer form of MCL1 regulates apoptosis, whereas the matrix form is important for mitochondrial function. It is not known which form is involved in regulating autophagy and mitophagy. Interestingly, we found that the outer form of MCL1 is more susceptible to degradation after a myocardial infarction, whereas the levels of MCL1 in the matrix remain intact. These data suggest that the inner mitochondrial form is somehow spared from the degradation process that targets the outer mitochondrial membrane isoform. This outer membrane isoform is accessible to E3 ubiquitin ligases like HUWE1/MULE and possibly PARK2. Interestingly, the outer MCL1 levels are restored within 24 h after the infarction, indicating that the heart takes effort to replenish MCL1 to ensure survival.

Our study has uncovered that MCL1 has additional critical functions in myocytes other than inhibiting apoptosis. The presence of MCL1 is critical for normal mitochondrial function, and to facilitate autophagy and mitophagy. Hence, loss of MCL1 leads to mitochondrial dysfunction and impaired autophagy. Since the affected myocytes are unable to clear damaged mitochondria via autophagy, these mitochondria start to accumulate, ATP levels become compromised, and necrotic cell death quickly ensues (Fig. 1). Degradation of MCL1 in heart may also compromise autophagy under stressed conditions when it is needed most, making cells more susceptible to cell death. Maintaining endogenous levels of MCL1 during these periods may support clearance of damaged organelles and improve cardiac outcomes. Finally, MCL1’s role in the heart may complicate efforts to antagonize the protein during chemotherapy. MCL1 antagonists have entered clinical trials for cancer therapy, and these drugs may lead to unexpected cardiotoxicity. To effectively employ BCL2 family antagonists as chemotherapeutics, mechanisms of MCL1 function will need to be better characterized in the heart.

Figure 1. Loss of MCL1 in adult myocytes results in mitochondrial dysfunction, defective PINK1-PARK2 signaling and impaired initiation of autophagy. As a result, there is an accumulation of dysfunctional mitochondria, which results in compromised ATP production and activation of necrotic cell death.

Thomas RL, Roberts DJ, Kubli DA, Lee Y, Quinsay MN, Owens JB, Fischer KM, Sussman MA, Miyamoto S, Gustafsson ÅB. Loss of MCL-1 leads to impaired autophagy and rapid development of heart failure. Genes Dev. 2013;27:1365–77. doi: 10.1101/gad.215871.113.

10.4161/auto.26168

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.