Protective Function of MCUb in Post Ischemic Remodeling: Getting at the Heart of the Calcium Control Conundrum

In the heart, rigorous regulation of intracellular calcium levels is integral to maintain cell survival and cardiac homeostasis. Calcium is a highly versatile signaling molecule that is involved in regulating many diverse processes, ranging from mitochondrial metabolism to cardiac contraction. Mitochondria are known to take up calcium and many mitochondrial enzymes in the tricarboxylic acid cycle are calcium-dependent¹. However, calcium also has a dark side where excess uptake can lead to destruction of mitochondria and activation of necrotic cell death². Mitochondrial calcium overload leads to opening of the mitochondrial permeability transition pore (mPTP) allowing for the influx of solutes and water into the matrix. This ultimately causes mitochondrial swelling and rupture with activation of necrotic cell death. Mitochondrial calcium overload and mPTP opening occur during myocardial ischemia/reperfusion and are major contributors to the injury². Not surprisingly, both mitochondrial calcium influx and efflux are highly regulated processes and an increased understanding of the mechanisms regulating these events could provide important avenues for therapeutic intervention to limit I/R injury in patients.

While it was recognized that mitochondria could take up calcium through a channel already in the 1960s, the molecular identity of the channel remained elusive for several decades. However, in 2010 the first regulator of the channel, mitochondrial Ca²⁺ uptake protein 1 (MICU1), was identified³, and in 2011 two groups simultaneously identified the mitochondrial calcium uniporter (MCU) as the pore forming component of the channel^{4, 5}. Once MICU1 and MCU had been identified, the field advanced very quickly and researchers promptly discovered a number of additional regulators of the calcium channel. It is currently known that the MCU complex is the key modulator of calcium transport into the mitochondrial matrix and that the pore forming complex is comprised of four major MCU subunits⁶. There are also a variety of other regulatory components that contribute to the monitoring of calcium levels. These include the essential MCU regulator (EMRE), mitochondrial calcium uptake proteins 1 and 2 (MICU1 and MICU2), and mitochondrial calcium uniporter regulator 1 (MCUR1)^7–9. In vitro studies have provided great insights into on the composition, regulation and function of the channel in cell lines. However, in vivo studies have yielded variable results regarding the physiological functions of MCU and its components in regulating mitochondrial calcium influx in tissues. For example, although cardiac mitochondria isolated from systemic Mcu knockout mice were found to be resistant to acute calcium uptake and mPTP opening, these mice were not protected against myocardial I/R injury¹⁰. Similarly, mice deficient in the regulatory protein EMRE also failed to show reduced infarcts after I/R injury, despite reduced calcium uptake in cardiac mitochondria¹¹. In contrast, cardiac-specific inducible deletion of Mcu in the adult mouse heart reduced mPTP opening and protected against ischemia/reperfusion injury^{12, 13}. Thus, given the complexity of MCU-mediated calcium influx into cardiac mitochondria in vivo with the existence of alternative uptake mechanisms, additional studies are urgently needed to dissect how calcium influx is regulated in the heart.

MCUb is a negative regulator of mitochondrial calcium uptake⁶ and in this issue of Circulation Research, Huo et al. offer fresh insights into to the physiological function of MCUb in the heart and provide persuasive evidence that the function of this protein is to limit post-infarct remodeling¹⁴. In this interesting study, the authors report that MCUb is undetectable in the mouse heart under normal conditions, but that its expression is rapidly induced in response to ischemic injury. This is in contrast to other cells and tissues that express MCUb at baseline⁶. Specifically, evaluation of MCUb levels in hearts of mice that had been subjected to 60 min of ischemia followed by reperfusion revealed that transcript and protein levels did not increase until 3 days after the ischemic injury, with levels even further increased after 7 days. The authors also found that the other components of the channel (MCU, EMRE, MICU1/2) were increased in response ischemic injury, but to a lesser extent than MCUb. This dynamic regulation of MCUb in the heart in response to ischemic injury is consistent with another recently published study by the Elrod group¹⁵. Additionally, the authors extended their findings to a model of permanent myocardial infarction and confirmed that MCUb was also increased in the heart at 3 and 7 days following injury. Intriguingly, they also discovered that MCUb was increased in hearts in a model of remote ischemic preconditioning from the hindlimb. These findings suggested that cardiac MCUb does not regulate mitochondrial calcium influx under baseline conditions. Rather, it is a stress activated protein that is upregulated in response to ischemic injury in an effort to limit mitochondrial calcium overload in cardiac myocytes.

The MCUb has been proposed to function as a dominant negative of MCU to alter the channel properties and reduce calcium channel activity (Figure)⁶. The MCU to MCUb ratio varies greatly between different tissues, suggesting variable channel activity. The fact that MCUb is undetectable in the heart indicates that the MCU channel might have higher baseline activity in cardiac mitochondria. Although this might allow for increased metabolic flexibility, it also creates a conundrum where cardiac mitochondria will be more sensitive to calcium overload and have a higher probability of mPTP opening. To further dissect the function of MCUb in the heart and determine whether manipulating MCUb levels could prevent excessive mitochondrial Ca²⁺ influx that occurs upon initiation of reperfusion after an ischemic episode, the authors generated cardiac-specific MCUb transgenic mice. As predicted, overexpression of MCUb in the heart led to decreased mitochondrial calcium uptake in both purified mitochondria and in intact permeabilized adult cardiomyocytes. The authors also observed that elevating levels of MCUb in the heart had no effect on mitochondrial oxygen consumption rate or overall baseline calcium content. These observations confirm the existence of an alternative calcium influx mechanism to prevent disruption of essential calcium dependent functions. Additionally, MCUb overexpression in the heart did not negatively affect cardiac structure or function but protected against acute myocardial I/R injury. The authors found that mitochondria from MCUb transgenic mice had decreased frequency of mPTP opening, which likely contributed to the reduced myocardial infarct size at 24 h post-I/R. These findings clearly demonstrate that reducing mitochondrial calcium uptake via MCUb overexpression can protect against acute I/R injury.

Figure 1. MCUb prevents mitochondrial calcium influx and protects the myocyte mitochondria against calcium overload and mPTP opening during post-infarct remodeling.

A. Ischemic injury causes calcium influx and overload through the MCU channel that leads to mPTP opening and cell death. B. MCUb protects mitochondria against calcium overload in response to ischemic injury by inhibiting MCU-mediated calcium uptake. This leads to decreased mPTP opening and reduced cell death. Ca²⁺ indicates calcium, mPTP indicates mitochondrial permeability transition pore, IMS indicates intermembrane space, and IMM indicates inner mitochondrial membrane.

However, the fact that MCUb is not detectable until 3 days post-injury suggested that the physiological role of the endogenous protein is not in preventing calcium uptake and mPTP opening upon initiation of reperfusion to reduce acute injury. Rather, it suggested that MCUb is more important in limiting post-ischemic remodeling that occurs days after the initial insult. To further investigate the physiological role of MCUb in the heart, the authors generated cardiac-specific MCUb knockout mice. The authors observed that deletion of Mcub in myocytes had no effect on mitochondrial calcium uptake and cardiac function, confirming that this protein does not regulate calcium influx in the heart under baseline conditions. More importantly, the authors discovered that wild type (WT) and Mcub-deficient hearts had similar in infarct size at 24 h of reperfusion, consistent with the notion that endogenous MCUb does not play a role in the acute injury phase. Given that endogenous MCUb is not induced until 3 days post-injury, the authors investigated the effect of Mcub-deficiency at later time point. Indeed, the Mcub^−/− mice had increased scar area and cardiac remodeling at 4 weeks after the injury compared to WT mice. The authors performed characterization of WT and Mcub^−/− cardiac mitochondria isolated from hearts 7 days post-I/R which confirmed that WT mitochondria had reduced calcium uptake compared to Mcub^−/−. The calcium uptake in Mcub^−/− mitochondria was similar to sham WT mitochondria, suggesting unrestricted calcium influx via the MCU-channel. These important findings clearly confirm that MCUb functions to limit post-ischemic remodeling, rather than acute injury.

In sum, the findings by Huo et al. represents a significant contribution to an exciting field. Specifically, the authors shed new light on the physiological function of MCUb in the heart. Utilizing a novel cardiac-specific inducible Mcub knockout mouse line, the authors elegantly demonstrate the importance of endogenous MCUb in limiting infarct expansion and post-infarct remodeling. Moreover, they also confirm that manipulation of MCUb levels in the heart prior to injury provides protection against calcium overload and acute I/R injury, validating the therapeutic potential of targeting mitochondrial calcium uptake. However, there are still many open questions that need to be explored. Future studies need to focus on further dissecting the molecular mechanism by which MCUb abrogates MCU-mediated calcium uptake into mitochondria. Also, the signaling pathways responsible for activating MCUb gene transcription in response to ischemic injury need to be identified. It is intriguing that IPC of the hind leg also leads to induction of MCUb in the heart and indicates that endocrine factors might be involved in promoting the gene expression under these conditions. Regardless of the unanswered questions, the study by Huo et al. provides exciting new insights into the physiological function of MCUb in the heart that will serve to drive future research forward.