Mitochondrial respiration is the most efficient way of producing energy in the form of adenosine triphosphate (ATP). In mitochondria, reducing equivalents produced by the tricarboxylic acid (TCA) cycle or coming from the β-oxidation of fatty acids (FAs) are oxidized by the multienzyme respiratory chain (complexes I–IV). Oxidation consumes oxygen to produce water, whereas protons are pumped out of mitochondrial matrix to create a proton gradient. The electrochemical gradient across the MIM drives ATP synthase to produce ATP. The key sources of reducing equivalents utilized in the heart are free FAs (FFAs), glucose, and lactate. They represent the majority of oxidative fuel directed into the mitochondria to enter TCA cycle.

Cellular energy demands dictate adaptive changes in energy production by the mitochondria. Depending on the challenge, mitochondria can change oxidative capacity modulating the activity of OXPHOS or biosynthesis of some of the OXPHOS components. In addition, the number and size of the organelles also can significantly be changed (see following section). Therefore, mitochondria, as major sources of ATP generation, are the targets for thyroid hormone-mediated regulation of cardiac metabolism [67, 68]. Goldenthal et al. [69] have demonstrated that major components of cardiac mitochondrial bioenergy-producing machinery are regulated by T₃.

It is well documented that cytochrome c oxidase enzyme is upregulated in response to T₃ treatment [70, 71]. In mammals, COX is composed of 13 polypeptides, ten of which are transcribed within the nucleus, and the remaining three ­subunits are products of the mitochondrial genome. In the myocardium, T₃ induces mitochondria-encoded COX1 expression, and it parallels the T₃-induced increases in COX activity. [69, 70] Levels and activity of COX1 (and also nuclear-encoded COX4) are significantly elevated shortly after in vitro treatment of neonatal rat cardiomyocytes with triiodothyronine (3–12 h) [69]. Surprisingly, while nuclear gene encoding another subunit of cytochrome c oxidase, COX5b, contains TRE in promotor region, it is not regulated by T₃ in the heart [70], suggesting a role for TR corepressors, limiting the induction of this gene in the heart. Instead of influencing the expression of COX5b, a unique nongenomic mechanism of interaction of thyroid hormones with this subunit has been described. In particular, diiodothyronines (and T₃ to lesser extent) have been shown to bind specifically to COX5b protein from bovine heart [72, 73]. This binding abolishes the allosteric inhibition of cytochrome c oxidase activity by ATP. Direct interaction of thyroid hormones with COX5b explains at the molecular level the short-term stimulatory action of thyroid hormones on mitochondrial respiration (Fig. 22.2). Yet another mechanism of regulation of cytochrome c ­oxidase by thyroid hormones acts through mitochondrial phospholipid, cardiolipin. According to Paradies et al. [74], cytochrome c oxidase activity is decreased in heart mitochondria from hypothyroid rats, and activity of this enzyme complex can be completely restored to the level of control rats by exogenously added cardiolipin.

Cytochrome c oxidase is not the only ETC component sensitive to thyroid hormones. For instance, gene encoding subunit 3 (ND3) of NADH dehydrogenase (also known as complex I) has been found to contain TRE. Transcriptional regulation of this gene by thyroid hormones in the heart has been demonstrated by Iglesias et al. [75]: level of ND3 mRNA is significantly diminished in hypothyroid rats compared to control animals. Remarkably, gene encoding ND3 is located in mtDNA, suggesting direct transcriptional regulation of ND3 by thyroid hormone via TRs identified inside the mitochondria (see previous section of this chapter). In vitro treatment of neonatal rat cardiomyocytes with triiodothyronine also elevates level and activity of ATP synthase (complex V) α-subunit (early response, 3–12 h) and activities of complex II and citrate synthase (late response, at 72 h) [69]. Apparently, long-term T₃ effect on mitochondria is complex, involving regulation of other mitochondrial proteins in addition to ETC. For example, it is accompanied by induction of ANT [69] and uncoupling protein (UCP) 3 [76], which cause an uncoupling in mitochondria [19, 77] with subsequent less efficient energy production in the cardiomyocyte (Fig. 22.2).

In addition to respiratory-chain components, proteins that connect cytosolic and mitochondrial metabolic pathways play a key role in the T₃-mediated modulation of energy metabolism. Thus, the expression of ANT type 2 mRNA is increased seven- to ninefold in rat heart within 12–48 h after T₃ application. These mRNA changes correlate with an increase of ANT protein, which explains the accelerated ADP/ATP exchange observed in mitochondria isolated from hyperthyroid rats [19]. Two myocardial transport pathways mediate transport of cytosolic reducing equivalents (in form of NADH) to the mitochondrial OXPHOS system. The malate/aspartate shuttle is the dominant NADH-transporting pathway in cardiomyocytes, whereas α-glycerophosphate shuttle is tenfold less active. The NADH shuttles have a potential to optimize bioenergy substrate utilization in hyperthyroid-induced cardiac hypertrophy when increased glycolytic flux takes place instead of utilization of FAs. The malate/aspartate shuttle capacity is significantly increased in ventricular mitochondria isolated from T₃-treated rats. In particular, protein levels of two components of the malate/aspartate shuttle system, mitochondrial malate dehydrogenase (mMDH) and aspartate/glutamate carrier (AGC), are found to be increased in the thyroid hormone-treated myocardium, suggesting that either protein could contribute to the increased capacity of the malate/aspartate shuttle. Taking into account that state of mMDH is the near equilibrium, the increased levels of AGC carrier seem to be responsible for the upregulation of the malate/aspartate shuttle in the ­thyroid hormone-treated cardiac mitochondria [78]. While regulation of mMDH gene by thyroid hormone is posttranscriptional (changes in mMDH protein levels are not accompanied by changes in mRNA), it is possible that the regulation of AGC gene expression is directly regulated by T₃. Transport of cytosolic NADH to the mitochondria via the glycerophosphate shuttle is very sensitive to T₃: mRNA of the FAD-linked mitochondrial α-glycerophosphate dehydrogenase (mα-GPDH), key component of this shuttle, is induced very fast (4–6 h) by T₃ in rat heart [19, 78, 79], and mα-GPDH protein levels increase in parallel [78]. These findings suggest that thyroid hormone directly stimulates nuclear transcription of the mα-GPDH gene (Fig. 22.2).

Key enzyme regulating glucose oxidation in mitochondria—pyruvate dehydrogenase complex (PDHC)—is also a target for thyroid hormone. Chronic T₃ supplementation in rats decreases activity of PDH. It has been shown that T₃ transcriptionally upregulates PDK4, which phosphorylates and inhibits PDHC [20, 21] (Fig. 22.2). It should be mentioned that a controversy exists between effects of T₃ on pyruvate oxidation in mitochondria and on pyruvate uptake by mitochondria: while oxidation of pyruvate is inhibited (via T₃-dependent regulation of PDK4/PDH), transport of this molecule into mitochondria increases [80]. Transport of pyruvate across mitochondrial membrane is mediated by mitochondrial pyruvate carrier, and Paradies and Ruggiero [80] have demonstrated that triiodothyronine significantly enhances this process. It is possible that in hyperthyroid cardiac mitochondria, increased pyruvate converts into oxaloacetate by mitochondrial pyruvate carboxylase (rather than oxidized via inhibited PDH) to support the oxidation (via TCA cycle) of acetyl coenzyme A (acetyl-CoA) derived from fatty acid oxidation (FAO). Interestingly, T₃-dependent enhancement of pyruvate uptake does not depend on the increase of pyruvate carrier expression but rather involves positive modulation of the pyruvate carrier activity by cardiolipin [80] (cardiolipin levels in mitochondria are known to increase in hyperthyroid rats—see below).

Another potential target for T₃-dependent modulation of substrate oxidation is direct regulation of lactate transport and oxidation in the mitochondria. Lactate generally supplies approximately 25% of myocardial oxidative substrate at normal physiological levels [81, 82]. It enters cardiomyocyte through the monocarboxylate transporter 1 (MCT1) [83] and is oxidized by lactate dehydrogenase within the mitochondria. Recent findings demonstrate colocalization of the MCT1, the MCT1-chaperone (CD147), and lactate dehydrogenase within mitochondrial reticulum of muscle cells [84]. T₃ promotes lactate exchange at the cell sarcolemma not via modulation of MCT1 expression but rather via changing its activity [85] and altering translocation of MCT1 and CD147 to the mitochondria.

The major cardiac functional changes induced by ­hypothyroidism include a drop in heart rate and a decrease in diastolic function, and these alterations are linked to energy metabolism in mitochondria affected by hypothyroidism. In particular, thyroid hormone alters FFA flux into TCA cycle via regulation of mitochondrial FAO. In fact, cardiac mitochondrial FAO is significantly depressed in hypothyroid rats [86], so the contribution of acetyl-CoA entering the TCA cycle via PDHC (lactate, glucose) increases in the hypothyroid state relative to that from FFAs and ketones [67]. The oxidation of long-chain FAs depends on their transport across the MIM mediated by the carnitine palmitoyltransferase (CPT)/carnitine–acylcarnitine translocase system. Depressed respiratory rates correlate with low FA translocation activity in cardiac mitochondria from hypothyroid rats, suggesting T₃-related changes in functional activity of FA-transporting system. In agreement with this hypothesis, it has been shown that systemic hypothyroidism in rats decreases expression of muscle-type CPTI (M-CPTI), a central component of the CPT/carnitine–acylcarnitine translocase system, associated with the MOM [67, 68]. In addition, Paradies et al. [86] have demonstrated 38% diminishing of carnitine–acylcarnitine exchange across the MIM of cardiac mitochondria from hypothyroid rats—a process catalyzed by another component of FA-transporting system, carnitine–acylcarnitine translocase (Fig. 22.2).

In the latter case, it has been suggested that the reduced mitochondrial translocase activity in hypothyroid rats could be due to the changes in cardiolipin levels in the lipid surrounding of the carnitine carrier molecule in the MIM. Cardiolipin locates almost exclusively in the MIM where it is biosynthesized [87–90] and has been reported to be specifically required for full functioning of the carnitine–acylcarnitine translocase (see also Chap. 3) [91, 92]. Importantly, cardiolipin content is markedly decreased in the cardiac MIM from hypothyroid rats and can be restored to the level of control rats by treatment of hypothyroid rats with T₃ [86]. Interestingly, it has been reported that the biosynthesis of cardiolipin in mitochondria is regulated by thyroid hormone via an alteration in the cardiolipin synthase activity [93, 94]. Thus, decrease of cardiolipin synthase activity and levels of cardiolipin may represent the molecular mechanism responsible for the decline in the activity of mitochondrial FA-transporting system under hypothyroid state.

Thyroid hormone influences uncoupling of substrate oxidation to ATP generation, which produces heat. An uncoupling is achieved by disturbing the proton gradient across the MIM by “proton leak” altering proton trafficking across the MIM or by covalent modification of the ADP/ATP ­translocator [95].

In rodents, UCP1, which belongs to the superfamily of mitochondrial anion carriers, mediates inducible proton leaks, and catecholamines and thyroid hormone regulate an expression of this protein. It is worth to note that this protein is unique to brown fat tissue, whereas other tissues express UCP2 and UCP3 that do not appear to be regulated by thyroid hormone [96–98]. Outside of brown fat, thyroid hormone regulates basal mitochondrial proton conductance by unknown mechanism [99].

Thyroid hormone plays an important role in postnatal metabolic adaptation of the myocardium. After birth, rise of the thyroid hormone prompts elevations in mRNA and protein level of ANT in cardiac mitochondria (Fig. 22.2). As a result, ADP/ATP exchange across the mitochondrial membrane increases, and cardiac mitochondrial respiration becomes ADP independent: increases in cardiac work are not accompanied by increases in ADP [22, 23].

The heart, which maintains a high oxidative capacity relative to other organs, must respond rapidly to changing energy requirements coming across physiologically or during stress conditions. One way to increase cardiac oxidative capacity and stimulate substrate oxidation pathways on demand is to elevate mitochondrial copy number (or higher mitochondrial mass) because the mitochondrion is a key regulator of the metabolic activity of the cell and is an important organelle in both production and degradation of free radicals. The process, by which new mitochondria are formed in the cell, is called mitochondrial biogenesis.

The expression of genes both in the nuclear and mitochondrial genome is required for mitochondrial production. The majority of mitochondrial protein is synthesized under control of the nuclear genome, while the mitochondrial genome encodes several components of ETC along with mitochondrial rRNA and tRNA. A major adaptive response of mitochondrial biogenesis results in more mitochondrial mass associated with increased glycolytic enzymes, oxidative phosphorylation, and ultimately with a greater mitochondrial metabolic capacity.

The master regulators of mitochondrial biogenesis appear to be the PGC-1 family of transcriptional coactivators, including PGC-1α, PGC-1β, and the PGC-related coactivator, PRC. PGC-1α, in particular, is thought to be a master regulator. It is known to coactivate NRF1 and NRF2 (also known as GABPA). The NRFs, in turn, activate TFAM, which is directly responsible for transcribing nuclear-encoded mitochondrial proteins. This includes both structural mitochondrial proteins as well as those involved in mtDNA transcription, translation, and repair.

The ability of thyroid hormone to increase the number of mitochondria in a cell is well known. In rats chronically treated with thyroid hormone, myocardial mitochondrial mass, mitochondrial respiration, and OXPHOS enzyme activities, mitochondrial protein synthesis and mtDNA levels increase, indicating overall increase in mitochondrial biogenesis [100]. Recent data show that T₃ coordinates cardiac mitochondrial and nuclear transcription required for the propagation of mitochondria [69, 100]. Thyroid hormone participates in the synthesis of nuclear-encoded mito­chondrial proteins contributing to substrate oxidation, the ETC, and oxidative phosphorylation (the vast majority of mitochondrial proteins) [101]. Studies using microarray techniques have identified approximately 4–8% of randomly selected genes to be regulated by T₃ in mammalian tissues [102–106], whereas only a limited number of genes have been proven to be directly regulated by TRs via TREs. Based on the fact that many T₃ target genes lack TREs in the regulatory elements together with the data of differential gene expression patterns after T₃ treatment (there are “early expression” and “late expression” T₃-inducible genes) [103, 104, 107], it appears multiple pathways are involved in T₃-mediated gene regulation. For instance, TRE-containing gene of mitochondrial glycerol-3-phosphate dehydrogenase is induced within the first hour after T₃ administration in hypothyroid rats in vivo [108]. In contrast, genes that have been induced late (>12 h) after treatment of hypothyroid rats with T₃ (ANT2, cytochromes c and c ₁, complex II, α- and β-subunits of mitochondrial ATP synthase, citrate synthase, TFAM) [19, 24, 25, 39, 69, 109] are regulated via indirect mechanisms.

Many nuclear genes encoding mitochondrial proteins that respond to T₃ in the process of mitochondrial biogenesis contain common DNA motifs in their promoters, which are recognition sites for nuclear regulatory factors, so they serve as intermediate mediators of T₃ action. For example, late T₃-inducible genes encoding cytochrome c and TFAM have functional sites for binding NRF1 [110–112]. NRF1 plays an essential function in mitochondrial biogenesis [113] and is endogenously regulated by T₃, probably via a TRE [104].

Transcription factors Specificity Protein 1 (Sp1) and Ying Yang 1 (YY1) have been demonstrated to be critical factors for their promoter activities of mitochondrial protein genes. Their target genes (ANT2, cytochromes c and c1, COX4, β-subunit of mitochondrial ATP synthase, TFAM) [110–112, 114–116] are regulated by T₃ in a late inducible manner, supporting the idea of Sp1 and YY1 as intermediate factors. However, it is currently unknown if Sp1 and YY1 are themselves regulated by T₃. Moreover, the DNA-binding proteins targeting the OXBOX/REBOX and Mt motifs in T₃-sensitive nuclear-encoded mitochondrial proteins (ANT, β-subunit of mitochondrial ATP synthase) are currently unknown [117–119].

Action of thyroid hormone on the expression of proteins during mitochondrial biogenesis can also be mediated via regulation of coactivators and corepressors of transcription factors—proteins that do not bind directly to DNA but interact with transcription factors and modulate their activity. For instance, PGC-1α is a potent coactivator of nuclear receptors (including TR) and NRF1 [33, 120] and has a profound influence on mitochondrial biogenesis and metabolic rate [33, 121]. T₃ promotes PGC-1 protein in cultured neonatal rat myocytes [69]. Importantly, these findings are in agreement with in vivo data: PGC-1α expression levels in hypothyroid rats increase 13-fold 6 h after administration of T₃ and remain high for at least 42 h [104]. Two other members of PGC-1 family, PRC and PGC-1β, show some similar binding properties to nuclear receptors [122–124], but it is currently unknown if they are regulated by T₃.

Also, level of global regulatory transcription factor PPARα increases in the myocardium of 15-day T₄-treated rats as well as in rat cardiomyocytes treated with T₃ in vitro [69, 100]. PPARα is implicated in the expression of enzymes catalyzing mitochondrial FAO [125] and an expression of UCP [126]. Thus, the parallel increase in mitochondrial biogenesis and PPARα level suggests the participation of PPARα in the mitochondrial biogenesis as a response to T₃.

It is important to highlight that by modulating PGC-1α [33] and TFAM [37–39], T₃ serves as a bigenomic coordinator, because these regulatory proteins are involved in mitochondrial transcription. Moreover, various thyroid hormone receptors have been demonstrated to import into mitochondria for binding and transactivation of the mitochondrial genome. In liver, truncated forms of the TRα1 [44, 45] and TRβ1 [47] specifically translocate into the mitochondrion, bind T₃, and stimulate generalized transcription of the mitochondrial genome via activation of genes in D-loop of mtDNA and expression of mitochondrial 12S and 16S rRNAs [44]. Full-length TRα1 as well as multiple truncate versions have been identified within cardiac mitochondria [45, 127, 128]. To conclude, the complex finely tuned communication network coordinates the expression of mitochondrial and nuclear genomes to produce new, fully functional mitochondria.

Thyroid hormone appears to target two mitochondrial enzymes involved in the biosynthesis of cardiolipin, one of the principle phospholipids in the mammalian heart. Cardiolipin is the major mitochondrial membrane lipid ­playing a vital role in mitochondrial function. Phos­phatidylglycerolphosphate synthase catalyzes formation of phosphatidylglycerolphosphate which after dephosphorylation serves as a substrate of cardiolipin synthase to synthesize cardiolipin. Administration of thyroxine has been shown to increase phosphatidylglycerol and cardiolipin content in rat ventricular mitochondrial fractions compared to controls (34% and 23% to 72%, respectively) [94, 129, 130]. The activities of phosphatidylglycerolphosphate synthase and cardiolipin synthase parallel T₃-induced changes in phospholipids, by increasing 3.5- and 1.5- to 2.8-fold, ­respectively [93, 94]. Thus, thyroid hormone-dependent stimulation of mitochondrial biogenesis includes increase in mitochondrial phospholipids in addition to mitochondrial proteins, RNA, and DNA. It is worth to note that T₃-dependent changes in cardiolipin concentration may underlie alterations in activities of several mitochondrial cardiolipin-sensitive protein systems, such as long-chain FA-transporting system [91, 92], ANT [131], cytochrome c oxidase [74, 132], and pyruvate and phosphate translocators [80, 92, 133].

Process of mitochondrial biogenesis, in addition to coordinated expression of mitochondrial proteins encoded by both the nuclear and mitochondrial genomes, synthesis of lipids, and insertion of nascent proteins in the lipid bilayer, also involves mitochondrial protein import pathway. Mitochondrial proteins that are encoded by nuclear genes are synthesized as precursor proteins on cytosolic polysomes. The precursors harbor mitochondria-targeting sequences, which allow cytosolic chaperones to escort them to mitochondria (see also Chap. 4). Once at the mitochondrion, the precursor interacts with import receptors, collectively called translocases. Translocases are located in both the outer and inner mitochondrial membranes (TOM and TIM, respectively). They guide the precursor in the mitochondrion, where the targeting signal is proteolytically cleaved to form a mature mitochondrial protein [134, 135]. The protein import in mitochondria is influenced by T₃ [136], and limited information exists regarding regulation of individual import machinery components by thyroid hormones. Thus, it has been shown that the 20-kDa translocase of the MOM (Tom20) and the matrix chaperone mtHsp70 are upregulated in response to T₃ [43, 137] with parallel increase in import of various mitochondrial enzymes [137]. Recently, altered thyroid status has been demonstrated to affect Tom34 levels in the myocardium; T₃-dependent changes in the mRNA expression of various components of the Tim machinery in vivo have also been observed [136].