Calcium Homeostasis in Mitochondria: Mechanisms, Diseases, and Therapeutic Approaches

“Powerhouse of the cell” and “battery of a car” are some common analogies you may have heard used to describe the mitochondria [1].

These analogies oversimplify the functions and role of mitochondria in cell physiology. In contrast to powerhouses with a single energy transformation purpose, mitochondria are multifaceted and multifunctional [1]. Additionally, mitochondria are pivotal in acting against any imminent threats posed against the cell by activating the cell danger response. Any disruption that causes mitochondrial dysfunction results in various diseases.

Calcium is an essential cation (Ca²⁺) in cells that acts as a second messenger in various signaling pathways, controlling essential cellular processes like growth, contraction, secretion, metabolism, and gene expression, as well as cell survival and death [2,3]. Precise regulation of mitochondrial calcium levels is crucial in various processes related to this organelle. If the balance of mitochondrial Ca²⁺ is disrupted, it can lead to distinct pathological conditions, varying depending on the type of cell affected. Intracellular Ca²⁺ levels are closely monitored by cells, detecting changes in amplitude, duration, frequency, and location [2-4]. As a result, they skillfully respond to maintain calcium homeostasis and safeguard the cell from harm.

Mitochondrial Calcium Signaling

Mitochondrial Ca²⁺ signaling and its homeostasis is a complex mechanism that can be summarized in the following steps [3,4]:

1. Cellular Stimulation [3]: Mitochondrial Ca²⁺ levels remain low during rest, and cellular stimulation increases cytoplasmic Ca²⁺ levels. Various factors, including neurotransmitters, hormones, or cellular stress, could be the reason for such stimulation.
2. Calcium Uptake [3]: The mitochondrial calcium uniporter (MCU) allows Ca²⁺ ions to enter the mitochondria in response to increased cytoplasmic calcium levels. The MCU is located in the inner mitochondrial membrane, and the uptake of Ca²⁺ is driven by the electrochemical potential across the mitochondrial membrane.
3. Buffering of Ca²⁺ in Mitochondrial Matrix [3]: Once Ca²⁺ ions enter the mitochondria, they are rapidly absorbed, increasing the mitochondrial calcium concentration. A buffering system in the mitochondria regulates and maintains Ca²⁺ levels within an optimal range.
4. Utilization of Ca²⁺ [3,4]: The increased Ca²⁺ concentration inside the mitochondria activates enzymes within the mitochondrial matrix, such as those involved in the tricarboxylic acid cycle, which is essential for cellular energy production.
5. Mitochondrial Calcium Extrusion [3,4]: Once mitochondrial Ca²⁺ completes its role in cellular processes, excess Ca²⁺ is expelled from the mitochondria. The extrusion of surplus Ca²⁺ is mediated by the Na⁺/Ca²⁺ antiporter (NCLX) located in the inner mitochondrial membrane. NCLX transports Ca²⁺ ions out of the mitochondria and back into the cytoplasm.
6. Calcium Homeostasis [3,4]: The uptake and release of Ca²⁺ by mitochondria play a crucial role in shaping calcium homeostasis, which influences cellular functions such as muscle contraction, cell growth, gene expression, and cell death processes.

Mitochondrial dysfunction led by calcium overload

As discussed above, higher calcium levels in the mitochondria increase reactive oxygen species (ROS) formation, can disrupt mitochondrial respiration, and result in cell death. Increased calcium levels in the mitochondria is also associated with multiple diseases:

1. Alzheimer’s Disease is characterized by accumulation of the amyloid beta protein, which leads to a decline in cognitive skills, especially those that control memory [3]. Amyloid beta increases the influx of Ca²⁺ in neuronal mitochondria, making them vulnerable to excitotoxicity and apoptosis [3-5]. As amyloid beta builds up, mitochondrial function declines [4,6]. An increase in cytosolic Ca²⁺ levels, oxidative stress, and reduced ATP synthesis all result in further Ca²⁺ overload and an even greater amount of oxidative stress as a result of amyloid beta accumulation [3,4,6,7].
2. Huntington’s Disease (HD) has been linked to an autosomal dominant CAG-triplet mutation in the huntingtin gene. HD is characterized by severe motor, cognitive, and psychiatric symptoms. Although the exact causes of HD are unknown, mitochondrial dysfunction plays a major role in its development [3,4]. There are conflicting results on the role of mitochondrial Ca²⁺ in HD [3]. According to some research, lower levels of mitochondrial Ca²⁺ is linked to disease progression, whereas others identified a link to an overload of mitochondrial Ca²⁺ [3,8-11]. Further studies are needed to make Ca²⁺ signaling a therapeutic target for HD.
3. Parkinson's Disease (PD) arises from a decrease in neurotransmitter dopamine and the accumulation of intraneuronal Lewy Bodies primarily composed of a-synuclein. PD is the most linked neurodegenerative disorder to mitochondrial dysfunction [3]. Numerous investigations have demonstrated that increased mitochondrial Ca²⁺ uptake and reduced export in PD cell models result in mitochondrial Ca²⁺ overload [3,4,12-14]. Ca²⁺ homeostasis in mitochondrial neurons is critical to the genesis of PD [3,15-17]. Modifying the uptake and efflux machinery of mitochondrial Ca²⁺ may offer effective treatment options.
4. Asthma is a chronic lung disease caused by muscle tightening and inflammation around the airways that makes breathing difficult. Mitochondrial dysfunction is associated with chronic asthma. In the presence of allergens, uptake of Ca²⁺ via MCU induces excess ROS production [18]. Excessive ROS production results in the death of lung epithelial cells, causing the loss of epithelial barrier function. Disruption of mitochondrial Ca²⁺ influx and increased cytosolic Ca²⁺ could increase airway hyperactivity, as observed in asthma [18]. Hence, MCU and other porters that can interfere with mitochondrial Ca²⁺ homeostasis are potential therapeutic targets for asthma.
5. Chronic Obstructive Pulmonary Disease (COPD) is characterized by chronic bronchitis, destruction of lung tissue, and airway hyperactivity. COPD development is strongly associated with mitochondrial dysfunction. Mitochondrial Ca²⁺ overload is observed in patients with COPD and cigarette smokers. Mitochondrial Ca²⁺ overload triggers mitophagy, which is a major cause of COPD. COPD patients are found to have a disrupted epithelial airway barrier in the lungs, and an overload of mitochondrial Ca²⁺ causes disruption of the tight junction in airway epithelial cells [18]. Hence, mitochondrial dysfunction caused by Ca²⁺ overload plays a significant role in the pathophysiology and development of COPD.
6. Friedreich Ataxia (FA) is caused by a mutation in the mitochondrial protein frataxin. FA is characterized by degeneration of the cerebellum, dorsal root ganglia, and cardiomyopathy. Frataxin overexpression in adipocytes leads to increased mitochondrial Ca²⁺ absorption through MCU, higher mitochondrial membrane potential, and increased ATP production [3,19]. Studies with a frataxin-deficient neuronal model isolated from rat dorsal root ganglia found that enhancing the mitochondrial calcium buffer capacity resulted in calcium dysregulation, caspase-3 activations, and apoptotic cell death, suggesting calcium homeostasis as a potential target for FA [3,4,19].
7. Idiopathic Pulmonary Fibrosis (IPF) is a serious and chronic disorder that affects the alveolar tissue of the lungs, which surrounds the air sacs. Although the exact reasons for IPF development are not yet understood, IPF patients show increased expression of MCU and mitochondrial Ca²⁺ overload compared to control subjects [18]. Studies found that non-functional MCU in macrophages exhibited significantly reduced levels of mitochondrial ROS and fatty acid oxidation [20,21]. As a result, they were resistant to the development of IPF. Targeting of MCU and mitochondrial calcium influx are a potential therapeutic target for IPF.
8. Lung Cancer is reported to be associated with mitochondrial calcium-led dysfunction [2,18].
9. Heart Failure is most commonly linked with mitochondrial calcium-led dysfunction. Overload of mitochondrial Ca²⁺ is associated with cardiovascular diseases like arrhythmia, hypertrophic cardiomyopathy, and postischemic heart failure [22-24].

Therapeutic approaches for mitochondrial calcium homeostasis

1. Nerve Growth Factors (NGF) have been demonstrated to aid in preserving and efficiently operating mitochondria in neurons. Proteins involved in mitochondrial calcium absorption, including the MCU, can be affected by NGF in terms of their expression and function [25]. The transport of calcium ions from the cytoplasm to the mitochondria is done by the MCU [19,26]. Increased MCU expression and activity have been related to NGF signaling, which promotes mitochondrial calcium absorption.
2. Vitamin C, also known as ascorbic acid, is a strong antioxidant, thus is capable of regulating mitochondrial function. The activity of ion channels involved in the transport of calcium across the mitochondrial membrane can be altered by vitamin C [3,19]. Vitamin C contributes to maintaining normal mitochondrial calcium levels by assisting in correct MCU function. It plays an important role in regulating neuronal mitochondrial calcium levels [3,25,26]. The antioxidant and mitochondrial support abilities of vitamin C protect neurons from oxidative stress brought on by calcium and promote overall neuronal health.
3. Vitamin E is a potent antioxidant that helps neutralize free radicals and ROS generated during cellular metabolism, including those within the mitochondria. It regulates calcium transporters like MCU. In cardiomyocyte models, vitamin E restored mitochondrial Ca²⁺ uptake [3,4,19].
4. Melatonin is a powerful antioxidant that scavenges free radicals and ROS produced during cellular metabolism. It protects mitochondrial integrity and function by lowering oxidative stress, which incidentally affects calcium control in these organelles [3]. Melatonin inhibits MCU expression, thereby increasing the cytoplasmic calcium overload [27]. NCLX expression is enhanced in the presence of melatonin, thus increasing the efflux of excess calcium from the mitochondria [27,28]. Additionally, mitochondria are also protected by the neuroprotective effects of melatonin. It can promote healthy mitochondria and lessen dysfunctional mitochondria, both of which are essential for efficient calcium management. If you'd like to learn more about melatonin and melatonin supplementation, you can read our previous article.
5. Methylene Blue (MB) is an antioxidant that plays a vital role in removing the ROS in mitochondria. MB is a known compound. for the enhancement of mitochondrial function. To learn more about MB and its role in mitochondria, read here. MB is also shown to impact the activity of MCU, hence regulating mitochondrial Ca²⁺ levels [29,30]. It has been investigated as a possible treatment for several diseases linked to mitochondrial malfunction because of its capacity to alter mitochondrial activity and calcium signaling [30]. Just Blue is 16 mg of pure pharmaceutical grade MB, a compound known as an electron cycler, donating electrons to the electron transport chain and scavenging the mitochondria and cytosol for free radicals and ROS. The result? Enhanced ATP production and enhanced antioxidant protection.

Conclusion

Mitochondria play a central role in both healthy and diseased states due to their involvement in oxidative metabolism. Most, if not all, mitochondrial functions rely on the presence of calcium ions. While we have understood the main components responsible for maintaining mitochondrial Ca²⁺ levels for over four decades, our comprehension of the molecular and mechanistic aspects of calcium dynamics is still in the early stages of development. A thorough understanding will help us decipher the molecular mechanism behind numerous diseases such as neurodegenerative, cardiovascular, and respiratory disorders. A better awareness will also help us develop potential therapeutic targets for mitochondrial Ca²⁺ overload.

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