Several studies have shown the importance of mitochondria and mPTP activity in cardioprotection and the pathophysiology of cardiomyopathies (Baines et al., 2005
; Burelle et al., 2010
). However, the role of mitochondrial biology in the developing heart is poorly understood, although a few reports indicate that mitochondria regulate cardiac stem cell and skeletal myocyte differentiation (Chung et al., 2007
; De Palma et al., 2010
; Yan et al., 2009
). Our results not only characterize the development of mitochondrial structure and function during embryonic cardiac myocyte differentiation, but further demonstrate that the mPTP lies upstream of changes in mitochondrial morphology, mitochondrial function, and myocyte differentiation. These results highlight changes in two mechanisms by which the mPTP may regulate cardiac myocyte differentiation: bioenergetics and redox signaling.
First, changes in mPTP activity may affect myocyte bioenergetics to regulate myocyte differentiation. In the developing heart, cardiac energetic demands must increase dramatically to match increasing cardiac performance required by embryonic growth (Conway et al., 2003
; Porter et al, 2011
), and closure of the mPTP may quickly increase ATP production by increasing the coupling of the electron transport chain and ATP synthase. In addition, Elrod and colleagues recently demonstrated that mitochondria from CyP-D null hearts maintain approximately 2-fold higher calcium content at baseline, resulting in greater mitochondrial activity and enhanced glucose utilization (Elrod et al., 2010
). Similar control of calcium levels in the mitochondrial matrix in the embryonic heart may account, in part, for the changes in ETC activity we observed during embryonic cardiac development and may result in increased ATP production. Finally, intrinsic changes in ETC complex composition and activity, such as we observed for complex I, may play a role in this process. Therefore, we propose that as the mPTP closes, mitochondrial function increases, and an increase in ATP production may allow myocytes to differentiate to a stage in which they are able to provide the contractile force needed to ensure adequate circulation and embryonic survival.
Second, our results suggest that mPTP activity regulates cellular redox signaling during myocyte differentiation, and, although the mechanisms by which mPTP regulates ROS remain to be determined, we speculate that complex I is involved, as it is a major source of mitochondrial ROS. We found that E9.5 myocytes were highly oxidized, but closure of the mPTP decreased ROS levels and enhanced myocyte differentiation. Antioxidant treatment also enhanced differentiation of E9.5 myocytes, while oxidant treatment significantly inhibited differentiation in both E9.5 and 13.5 myocytes. These data suggest a link between the mPTP, redox signaling, and myocyte differentiation, which we tested by combining agents that affected the mPTP and oxidative state. Upon treatment of E9.5 myocytes with the antioxidant concurrent with the mPTP-inducing CAT treatment, myocyte differentiation remained enhanced. In contrast, when E9.5 myocytes were treated with oxidant concurrent with the mPTP-closing BKA treatment, myocyte differentiation remained inhibited. Together, these observations suggest that the control of ROS levels could be a mechanism by which the mPTP regulates differentiation between E9.5 and 11.5. Consistent with this idea are recent studies, which show that embryonic stem cells also rely on redox signaling during differentiation into cardiac myocytes (Buggisch et al., 2007
Opening of the mPTP in the early embryonic heart may play a protective role
According to the data presented in Krishnan (2008)
, between E8.5 and 10 the pO2
in the mouse heart is lower than 10 mmHg. It is likely that this is lower than what is observed in the rest of the embryo at that age due both to a lack of effective placental circulation and increased energy requirements of the heart, which has begun to beat and circulate blood. After E10, the pO2
of the heart is higher due to the initiation of effective placental circulation. Therefore, major changes occur in the physiology of the heart between E9.5 and 11.5 as oxygen supply and energy demands increase.
The purpose of this mPTP-mediated delay of differentiation in early development may be, in part, to compensate for physiological changes that have yet to occur. The increase in energy demands likely requires dramatic maturation of mitochondrial function. However, the low oxygen supply at E9.5 may inhibit this maturation; e.g., these very immature myocytes have few mitochondria with immature mitochondrial structure and function as well as high oxidative stress, despite the low oxygen tension. This paradoxically high oxidative stress may be due to an immature ETC, decreased anti-oxidant defenses that are controlled by mitochondria (Aon et al., 2010
), or a yet undetermined mechanism. We hypothesize that the high ROS levels and low ATP production from immature mitochondria may prevent further myocyte differentiation in order to protect the heart during this period of low oxygen supply. Yet, as the placental circulation is established and oxygen supply increases, mitochondria mature, leading to decreased oxidative stress and increased ATP production. This maturation allows myocytes to differentiate directly via redox signaling and indirectly by increasing available energy, as discussed above.
If this is the case, then changes in the function of the mPTP may cause abnormal differentiation of cardiac myocytes and disrupt cardiac morphogenesis. Although it was initially reported that CyP-D null mice have no cardiac phenotype and survive gestation and have a normal lifespan (Baines et al., 2005
; Basso et al., 2005
; Nakagawa et al., 2005
), we observed “premature” mitochondrial maturation and myocyte differentiation in E9.5 CyP-D null myocytes compared to wild-type controls. In contrast we found that myocytes from older CyP-D null hearts were indistinguishable from their WT controls suggesting a critical period between E9.5 and 11.5 for mPTP closure that may not necessarily be pathologic. However, the abnormal variation in developmental stages in occasional litters of CyP-D mice we have observed may also indicate subtle pathology in the early embryo. Moreover, a recent paper shows that CyP-D mice do have abnormal cardiac physiology, particularly when placed under stress (Elrod et al., 2010
). Therefore, deletion of CyP-D may not be a completely benign.
In contrast, although deletion of CyP-D and closure of the mPTP, with the resulting mild acceleration of cardiac myocyte differentiation, is generally tolerated, we postulate that the converse phenomenon, sustained opening of the mPTP, would be much more devastating. If this were to occur, then mitochondrial maturation and myocyte differentiation would be inhibited leading to an inability of the developing heart to meet the demands of the growing embryo. Future experiments that manipulate mPTP opening and closing via changes in CyP-D and other putative components such as ANT may answer these questions.
Summary and future directions
In conclusion, these results demonstrate that mPTP function regulates myocyte differentiation directly via redox signaling pathways and suggest that these changes in mPTP function, mitochondrial maturation, and ROS levels are a mechanism that protects the embryonic heart during a period of increasing energy demands but low oxygen supply. Therefore, these data may have clinical implications by suggesting that some cardiomyopathies and congenital heart defects may be due to disruption of myocyte differentiation secondary to defects in mPTP activity, mitochondrial maturation, and redox biology. Thus, not only does this research define the important role of mitochondrial maturation and redox state in the developing heart, but it also may lead to therapeutic innovations to promote mPTP closure or decrease oxidative stress to accelerate myocyte differentiation in cardiac development, cardiomyopathies, and cardiac regeneration. Finally, these data suggest that increased IMM permeability is physiologic in the developing heart and that CyP-D activity is a mechanism by which mPTP is regulated in the early heart. Future studies should determine the mechanisms that control mPTP activity in the developing myocyte and further characterize the redox signaling pathways that lie downstream of mitochondrial function to control myocyte differentiation in the developing heart.