Mitochondria and heart failure: new insights into an energetic problem

Abstract

Cardiac mitochondria are powerful organelles supplying energy to support the high adenosine triphosphate (ATP) consumption of the beating heart. The progression of HF (HF) is characterized by diminished energy metabolism, calcium mishandling, reactive oxygen species (ROS) generation and apoptotic cell death. Although the etiologies of HF are multifactoral, many of the changes of HF are associated with cardiac mitochondrial dysfunction either directly or indirectly. A number of studies have established the role of calcium mishandling and reduced ATP production in mitochondrial dysfunction in HF. More recent work has contributed to our understanding of the role of ROS and proapoptotic protein release by the mitochondria in HF. New interest has been generated in mitochondria by the relatively recent identification of the processes of fusion and fission, which are critical to the maintenance of healthy mitochondria. Fission and fusion also have significant roles in apoptosis. Other studies have shown that estrogen has important functions in the mitochondria, including regulation of mitochondrial gene expression. Aging alone contributes to the development of HF through multiple mechanisms. These new insights into HF have implications for our understanding of this important disease, and will be reviewed here.

Overview

Heart failure (HF) is a complex disease that presents ongoing challenges as cardiac function declines. The etiologies of systolic HF are multifactoral, but once established HF progresses steadily, although this can be slowed by therapy. The progression of HF is associated with diminished energy metabolism, calcium mishandling, reactive oxygen species (ROS) generation and apoptotic cell death, as reviewed in a number of excellent publications.^1–9 Many of the cellular abnormalities associated with HF involve the mitochondria (Figure 1). Although mitochondria had been extensively investigated in the past, interest in this area had lapsed until the development of new techniques and new findings generated renewed interest in this important organelle. This review will provide an overview of mitochondrial function in the normal and failing heart, and then focus on novel aspects of mitochondrial dysfunction in HF, which may lead to new therapeutic options. Many studies of mitochondria have looked at the effects of ischemia, an important cause of HF. As these studies are pertinent to our understanding of mitochondrial changes in HF, they are included in this review.

Figure 1.

Diagram summarizing involved signaling pathways affected by HF. Mitochondrial components including those regulating ATP production, calcium handling, ROS generation, fusion/fission balancing, as well as pro/anti-apoptotic proteins are involved in the progression of HF. Estrogen is also involved in affecting the mitochondria in HF both directly and indirectly.

Changes of mitochondrial energy synthesis and oxidative stress in heart failure

ATP synthesis

An adequate adenosine triphosphate (ATP) supply is essential for maintaining cardiac function. Numerous studies have identified decreased high energy phosphate levels and flux as consistent features of HF. HF changes all components of cardiac energetics: substrate utilization, ATP production, and ATP transfer to the cardiac contractile apparatus.^{3, 5} Based on in vivo measurements by phosphorus magnetic resonance spectroscopy, ATP levels of patients with dilated cardiomyopathy (DCM) were reduced 35%.¹⁰ Diminished ATP levels were also found in a rat coronary ligation model of HF.¹¹ Similarly, a progressive decrease in ATP occurred during the development of HF in a canine model.¹² Mitochondrial structural abnormalities and decreased citrate synthase activity, provided further evidence of impaired mitochondrial function.¹³ ATP synthase activity was decreased 50% in Doberman pinschers with inherited dilated cardiomyopathy.¹⁴ Recently, Rosca et al. found that the ATP synthase activity in skeletal muscle mitochondria was also reduced in canine pacing-induced model of HF, suggesting systemic changes.¹⁵ Thus, in multiple models there is a clear decrease in ATP synthesis capacity and a decrease in overall ATP levels in HF (Figure 1 summarizes the changes and pathways discussed in this review).

As mitochondria are the major source of ATP in the heart and ATP is reduced in HF, investigations have focused on changes in mitochondrial function in HF. The electron transport chain (ETC) is an essential component of ATP production, and is localized to the mitochondrial cristae on the inner mitochondrial membrane. Defects have been reported in the individual ETC complexes in HF.^{16, 17} Sharov et al. showed that mitochondrial respiratory function is abnormal in both dogs and humans with chronic HF. In the myocardium of dogs with HF, the state 3 respiratory rate (V_ADP, after addition of ADP) was reduced 50–60% compared to normal.¹⁸ In explanted failing human hearts (ischemic and idiopathic) the respiratory control ratio, V_ADP/V_AT (respiration after the addition of atractyloside), was decreased compared to normal hearts.¹⁷ Rosca et al.¹⁹ identified a defect in oxidative phosphorylation within the ETC in HF. They found that the amount of supercomplex, consisting of complex I/complex III dimer/complex IV, which is considered the major form of the respirasome and essential for oxidative phosphorylation, was decreased. Cardiolipin (CL), a key inner mitochondrial membrane phospholipid involved in the integrity and flux of the ETC, also was depleted in HF. Dietary interventions may restore CL to its linoleic acid-rich form and improve cardiac function by redirecting the remodeling process.²⁰ Coenzyme Q10 (CoQ1O) is essential for electron transport within the mitochondria, and hence for ATP generation and cellular energy production. Molyneux et al. recently demonstrated that plasma levels of CoQ1O are an independent predictor of survival in a cohort of 236 patients with chronic HF followed for a median of 2.69 years.²¹ This is consistent with previous studies which have shown myocardial CoQ10 depletion in CHF,^{22, 23} supporting CoQ10 as a possible adjunctive therapy in CHF.

Grover et al. demonstrated an increase in ATP hydrolysis by the mitochondrial F(1)F(0)-ATPase in ischemic myocardium,²⁴ which is hypothesized to contribute to the overall depletion of the cellular ATP pool during ischemia. Selective inhibition of ATP hydrolase activity can protect failing myocardium. The adenine nucleotide translocator (ANT) and F1-ATPase, which are located in the mitochondrial membrane, respectively regulate mitochondrial adenosine 5’-diphosphate (ADP)/ATP exchange and ADP-phosphorylation, key components of high-energy phosphate metabolism. Deficits in myocardial ANT transcript levels, protein content or carrier activity have been reported in several models of CHF and myocardial stunning.^{25, 26} Histological and ultrastructural examination of heart muscle from ANT isoform 1 null mice revealed cardiac hypertrophy with mitochondrial proliferation, while mitochondria isolated from the skeletal muscle exhibited a severe defect in coupled respiration.²⁶ ANT isoform1 and the beta subunit of the F1-ATPase proteins were found to be significantly decreased in a HF model in Yorkshire swine (LAD ligation).²⁷ Moreover, the transcriptional regulator, PGC-1α, which controls mitochondrial biogenesis and the synthesis of the metabolic pathways for ATP synthesis, was down-regulated in both failing and hypertrophied hearts.^{28, 29}

Chronic ischemia is an important cause of HF. Altered mitochondrial uncoupling, affecting total ATP synthesis rates, could be an important mechanism to explain decreased cardiac energy.³⁰ Downregulation of uncoupling proteins (UCPs), especially cardiac expressed UCP3, in the setting of ischemia-induced HF was reported, together with decreased UCP3 mRNA levels compared to their normoxic controls.^{8, 31, 32} These results suggest that UCP3 might be involved in the decreased energy status in the failing heart. Brennan et al. using a low concentration of FCCP, a potent uncoupler of oxidative phosphorylation in mitochondria, significantly improved post-ischemic functional recovery via a ROS-dependent pathway without mitochondrial K_ATP channel activation, suggesting a role for mitochondrial uncoupling in failing hearts.³³ However, other experiments using rat models have shown that chronic hypoxia reduced oxygen consumption and ATP synthesis rate in isolated mitochondria, and here the mitochondrial K-ATP channel played a critical role.^{34, 35} Thus, these findings suggest a widespread depression of the mitochondrial ATP synthesis pathway during ischemia/hypoxia, which likely contributes to the process of HF.

ROS generation

ROS have direct effects on cellular structure and function and may be integral signaling intermediates in many pathways, including myocardial remodeling.³⁶ Mitochondria are thought to be both a major source of ROS, as well as a major target for ROS damage. Numerous experimental and clinical studies have shown increased generation of ROS in HF.^37–39 Importantly, Tsutsui et al. demonstrated that the activity of SOD, catalase, and GSHPx were not decreased in failing hearts, indicating that oxidative stress in HF is primarily due to the enhancement of pro-oxidant generation rather than to the decline in antioxidant defenses.⁴⁰ The mitochondrial ETC is an important source of ROS in HF.^{41, 42} ROS are generated in the ETC at complexes I and III. There is a positive correlation between mitochondrial membrane potential and production of ROS by ETC. Ide et al.⁴³ demonstrated that mitochondrial ETC I is a potential source of ROS in the failing myocardium. The inhibition of electron transport at the sites of complex I and complex III in the normal submitochondrial particles resulted in a significant production of ROS as demonstrated in studies using ESR spectroscopy with 5,5’-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap. Although increased ROS generation, rather than decreased anti-oxidants, is thought to be a contributing factor in HF development, reduction in anti-oxidant protein expression can provide proof of principle. In a transgenic Mn-SOD-deficient mice model loss of manganese superoxide dismutase led to progressive congestive HF with specific molecular defects in mitochondrial respiration.⁴⁴ In isolated cardiac myocytes, a subtle increase in ROS caused by partial inhibition of SOD resulted in a phenotype characterized by hypertrophy and apoptosis.⁴⁵ Mitochondrial ROS may be particularly prone to trigger apoptosis through activation of the mitochondrial permeability pore transition, release of cytochrome c, and activation of effector enzymes in apoptotic signaling.⁴⁶ Recent studies with isolated cardiomyocytes have implicated the effects of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), in the generation of ROS in mitochondria, contributing to the process of HF.^{47, 48} Inhibition of ETC complex I and mitochondrial uncouplers have been shown to reduce ischemia-reperfusion injury; it is proposed that this protection is mediated by reduction in ROS during ischemia.^{49, 50} Thus, ROS are major contributors to mitochondrial dysfunction in HF.

Mitochondrial calcium and heart failure

HF is also characterized by dysregulation of Ca2⁺ homeostasis within the myocyte.^51–53 Mitochondrial Ca2⁺ uptake from the cytosol controls the rate of energy production, which is diminished in HF.⁵⁴ The mitochondrial Ca2⁺ uniporter and the mitochondrial Na⁺/Ca2⁺ exchanger (mNCX) are the major pathways for Ca2⁺ transport across the cardiac mitochondrial inner membrane.⁵⁵ It is likely that various factors such as pH, ATP:ADP ratio, ATP:AMP ratio, and K⁺ :Na⁺ ratio, in addition to mitochondrial structural damage, play an important role in inhibiting mitochondrial calcium transport in failing hearts.⁵⁶ In failing human cardiomyocytes, the kinetics of mitochondrial Ca2⁺ uptake are altered, and increased resting [Na⁺]_i in HF contributes to impaired mitochondrial Ca2⁺ uptake.⁵⁷ Simulated ischemia studies in which the oxygen and glucose were excluded, show a rise in mitochondrial Ca2⁺.^{34, 58–61} Griffiths et al. observed that the rise in mitochondrial Ca2⁺ during ischemia was inhibited by clonazepam, an inhibitor of mitochondrial Na⁺/Ca2⁺ exchanger (NCX), suggesting a role for mitochondrial NCX operating in the reverse mode to increase mitochondrial Ca2⁺.⁵⁸ Ru360, a specific mitochondrial calcium uptake inhibitor, improves cardiac post-ischemic functional recovery in rats in vivo.⁶² Interestingly, by directly patch clamping the inner membrane of mitochondria from non-failing and failing human hearts, Michels et al. identified that the Ca2⁺ uptake is mediated by 2 previously unknown human mitochondrial Ca2⁺-selective channels, referred to as mCa1 and mCa2, which are functionally impaired in HF.⁶³ Mitochondrial Ca2⁺ overload following ischemic injury, can promote opening of mitochondrial permeability transition pore (MPTP), contributing to the process of necrosis and/or apoptosis that lead to HF.^64–67 In animal models, inhibition of MPTP opening by either cyclosporin A (CsA) or genetic ablation of cyclophilin D provides strong protection from both reperfusion injury and congestive HF. Griffiths et al. reported CsA, protected cells subjected to simulated ischemia and reperfusion; protection was defined as ability to recover rod shape and respond to electrical stimulation.⁶⁸ By using transgenic mice with enhanced sarcolemmal L-type Ca2⁺ channel activity, Nakayama et al. show that loss of cyclophilin D, a regulator of MPTP, blocked Ca2⁺ influx-induced necrosis of myocytes, HF, and isoproterenol-induced premature death.⁵¹ Mitochondrial membrane potential, cytochrome c oxidase levels, oxidase-dependent respiration and the rate of ATP synthesis were decreased in HF compared to normal controls, and these changes were reversed by treating for 24 hours with CsA.⁶⁹ These observations suggest that MPTP opening contributes to the loss of mitochondrial function observed in the failing hearts and inhibition of MPTP opening represents a potential therapeutic target for the treatment of HF. However, controversy remains over the role of ROS vs. calcium and currently it is favored that ROS, but not Ca2⁺ overload, trigger pH and mitochondrial permeability transition-dependent death of adult rat myocytes after ischemia-reperfusion.⁷⁰

Mitchondria, apoptosis and heart failure

Apoptosis is an important mechanism of myocyte loss in the progression of HF.^{1, 2, 71–75} Here we specifically review the intrinsic mitochondrial apoptotic pathways. Endonuclease G (EndoG), a nuclear-encoded endonuclease, is released from the mitochondria with injury and can cleave chromosomal DNA, contributing to apoptosis in HF.⁷⁶ AIF, a mitochondrial flavoprotein, has both oxidoreductase and apoptosis-inducing activities.⁷⁷ The subcellular translocation of AIF from mitochondria to the cytosol and nucleus was demonstrated by immunofluorescent analysis using confocal microscopy. However, by RT-PCR, AIF and EndoG mRNA were decreased, rather than elevated in HF.⁷⁸ The critical role of cytochrome c release into the cytoplasm leading to activation of caspase 3 is well established.^79–82 Chang et al. in an important set of experiments found that serum response factor (SRF), a central cardiac transcription factor, was a cleavage target for modestly activated caspase 3 in HF. This cleavage of SRF may play a dominant inhibitory role in propelling hearts toward failure by mechanisms other than apoptosis.⁸³ Thus, mitochondrial pro-apoptotic factors can potentially cause cell damage through more than one mechanism.

Data from ultrastructural and biochemical analysis of explanted human hearts support the constitutive activation of a cytochrome c-mediated apoptotic cascade in human cardiomyopathy.⁷⁴ Wu et al. recently found that ischemic stress in vivo and oxidative stress in vitro leads to up-regulation of COX III, followed by down-regulation of COX I expression, impaired COX oxidative activity, and increased DNA fragmentation and caspase 3 activation. Therefore, up-regulation of COX III may also contribute to the increased apoptosis following MI and subsequent HF.⁷³

The Bcl-2 family proteins are important regulators of the mitochondrial apoptotic pathway.⁸⁴ This family is composed of both pro- and anti-apoptotic proteins that share up to four conserved regions known as Bcl-2 homology (BH) domains. Anti-apoptotic members such as Bcl-2 and Bcl-X_L contain all four subtypes of BH domains, and promote cell survival by inhibiting the function of the pro-apoptotic Bcl-2 proteins. The pro-apoptotic members can be separated into two structurally distinct subfamilies. The multidomain proteins (Bax and Bak) share three BH regions and lack the BH4 domain. They are structurally similar to the anti-apoptotic proteins.^84–86 Hearts from Bax deficient mice had reduced mitochondrial damage and decreased infarct size after I/R compared to wild type, implicating Bax as a major player in mitochondrial dysfunction in I/R.⁸⁷ An increase in Bax and decrease in Bcl-2 has been reported in the pacing-induced model of HF.⁸⁸ Moreover, over expression of Bcl-X_L inhibited Bax translocation from the cytosol to the mitochondria, reduced cytochrome c release from mitochondria and decreased apoptosis after ischemia injury.⁸⁹ The activated truncated Bid (tBid) can also translocate to the mitochondria causing activation of Bax/Bak and release of cytochrome c into the cytosol.⁹⁰ Bnip3 and Nix/Bnip3L also have been associated with mitochondrial dysfunction and cell death in the heart. Bnip3 has been shown to contribute to I/R injury via activation of Bax.^{91, 92} Bnip3 was found to be upregulated in failing hearts, and subcellular fractionation experiments demonstrated that Bnip3 integrated into the mitochondrial membranes during hypoxia.⁹³ These findings illustrate the major role the Bcl-2 family plays in the apoptotic process in heart.

Interestingly, transgene-positive mice expressing high levels of either wild-type or activated RhoA (a small G-protein regulating the myocardial actin cytoskeleton) showed only a modest degree of cardiac hypertrophy, but rapidly developed dilated cardiomyopathy and HF likely through Bax mediated apoptosis.⁹⁴ In cardiomyocytes adenoviral overexpression of activated RhoA induces hypertrophy, which transitions over time to apoptosis by up-regulating Bax. This response was blocked by dominant negative p53 mutant, indicating that RhoA/Rho kinase activation up-regulated Bax through p53 to induce the mitochondrial death pathway and apoptosis.⁹⁵ Thus, RhoA activation alone can lead to apoptosis via Bax.

HSP60, primarily a mitochondrial protein, can interact with Bax and the mitochondrial apoptotic pathway in HF.^{72, 96–98} HSPs have a complex role in apoptosis, but primarily are anti-apoptotic. Pretreatment with heat can protect against apoptosis.⁹⁹ Overexpression of HSP60 prevented apoptosis in cardiac cells ¹⁰⁰ while reduction of endogenous HSP60 with antisense treatment precipitated apoptosis,¹⁰¹ in contrast, it has been found that HSP60 accelerated activation of caspase 3 during apoptosis in established cancer cell lines.¹⁰² In normal cardiac myocytes, we have shown that cytosolic HSP60 complexes with Bax and Bak by co-immunoprecipitation experiments ¹⁰¹ and by immuno-EM.⁷² Shan et al. confirmed the association of Bax with HSP60, and found that Bcl-XL also complexed with HSP60 in the normal heart, but not Bcl-2.⁹⁸ We also have demonstrated that both HSP60 mRNA and protein were increased in failing human and rat hearts,^{71, 96, 103} and this upregulation was mediated by NFκB.⁹⁶ In HF, HSP60 was present on the plasma membrane, and this was associated with apoptosis. To further investigate this, we treated isolated cardiac myocytes with HSP60. Caspase 3 activation and DNA fragmentation were found after treatment with recombinant HSP60, and this could be prevented by heat denaturation of the protein.¹⁰⁴ Apoptosis was mediated through activation of TLR4. Thus, the role of HSP60 in apoptosis may be both pro- and anti-apoptotic, depending on the cell type, HSP60 localization, and provoking stimulus for apoptosis.

The intrinsic, mitochondrial, apoptosis pathway is activated in HF and there is chronic low level activation of caspase 3. Caspase 3 activation may not only lead to apoptosis, but to changes in gene expression in the absence of apoptosis. The extrinsic pathway may also contribute to apoptotic cell death in HF via activation of this pathway by TNF, which is present in the plasma in HF. Extracellular HSP60 may contribute to myocyte loss through apoptosis.

Changes of mitochondrial dynamics in failing hearts

Mitochondrial fusion/fission

In contrast to the common view that mitochondria are static factories producing energy, mitochondria are dynamic organelles that continuously divide and fuse within the cell ^{105, 106} (Figure 2). At steady state the frequencies of fusion and fission events are balanced, maintaining the overall morphology/function of the mitochondria.¹⁰⁷ Abnormalities in fission and fusion can lead to apoptosis.^108–113 Proteins controlling mitochondrial fission in mammalian cells include Drp1 (DNM1 in yeast) ¹¹⁴ and Fis1.¹¹¹ Inhibition of Drp1’s GTPase activity with a dominant negative protein defective in GTP binding (Drp1K38A) results in elongated mitochondria in mammalian cells.^{109, 110, 115} Drp1 exists primarily in the cytoplasm but also associates into complexes on the outer surface of mitochondria at sites of organelle fission.¹¹⁶ Human Fis1 (hFis1) contains a tetratricopeptide repeat motif ¹¹⁷ facing the cytoplasm, and is anchored in the outer mitochondrial membrane via a C-terminal hydrophobic tail.¹¹¹ hFis1 circumscribes the outer surface of mitochondria and is not localized specifically to mitochondrial scission sites.^{111, 117} RNA interference (RNAi) mediated reduction of mammalian Fis1 resulted in mitochondrial elongation.¹¹² Mitochondrial fusion in mammalian cells is controlled by the large GTPases Mfn1,^{115, 118} Mfn2,^{115, 118, 119} and optic atrophy (Opa) 1 (MGM1 in yeast).^120–122 Elimination of any of these proteins induces mitochondrial fragmentation.^{120, 123} Mutations in Opa1 cause dominant optic atrophy,^{124, 125} while Charcot-Marie-Tooth disease type 2 is associated with mutations in mitofusins, among others ^126–193 (Table I). During apoptosis, the mitochondrial network fragments, resulting in smaller and more numerous mitochondria.^{109, 120, 128–130} It has been well reported that unbalanced mitochondrial fusion/fission interacted with the apoptotic pathways leading to cell death. Increased fission, decreased fusion, or both can induce caspase activation,¹⁰⁸ Bax translocation to mitochondria and cytochrome c release,¹²⁹ while Bak could also regulate apoptosis by interacting with mitofusins.¹³¹ These studies suggest a mechanistic link between mitochondrial morphology and apoptosis. Drp1 binding to mitochondria is increased during apoptosis,^{109, 132} and interestingly, the Drp1 foci at mitochondrial scission sites colocalize with foci of Bax.^128–130 Inhibition of Drp1 GTPase activity with a dominant negative protein (Drp1^K38A) prevents the mitochondrial fragmentation seen during apoptosis and delays the process of cell death,^{109, 128} suggesting that mitochondrial fission is a required step in apoptosis. Overexpression of hFis1 has been reported to induce apoptosis,¹³³ again implicating the involvement of mitochondrial fission in apoptosis. Down-regulation of Opal expression in cells by RNAi results in spontaneous cell apoptosis.¹²⁰ Recently we found that overexpression of Opa1 increased mitochondrial elongation, but could not prevent ischemia-induced apoptosis, suggesting that OPA1 levels are tightly controlled in the cell, and perturbation of these levels is not protective.¹³⁴ Thus, the balance of fission and fusion proteins is critical to the maintenance of cell homeostasis and the prevention of apoptosis.

Figure 2.

Models describing mitochondrial fusion/fission and involved proteins. Mfn1/2 facilitates mitochondrial outer membrane (MOM) tethering and OPA1 facilitates mitochondrial inner membrane (MIM) tethering to complete the mitochondrial fusion. Drp1 and Fis1 work together to complete the mitochondrial fission.

Table I.

Mammalian mitochondrial fusion/fission associated proteins.

Mammals	Yeast	Localization	Links to diseases
Mitochondrial fusion
Mfn1/Mfn2	Fzo1p	Mitochondrial outer membrane	Decreased in AD 191; Mfn2 mutated in CMT type 2A disease; 126, 127 Mfn2 is repressed in obesity 192
OPA1	Mgm1p	Mitochondrial inner membrane	Mutated in autosomal dominant optic atrophy; 125 decreases in HF; 134 decreased in AD 191
Mitochondrial fission
Drp1	Dnm1p	Cytosol/mitochondrial association	Increased in HF; 134 decreased AD 191, 193
Fis1	Fis1p	Mitochondrial outer membrane	Increased in AD 191

AD: Alzheimer’s disease; HF: heart failure.

We have recently reported that OPA1 ¹³⁴ is decreased in both human and rat HF. Furthermore, reduction in OPA1 increased apoptosis both at baseline and after simulated ischemia, via cytochrome c release from mitochondria in H9c2 cells. Electron microscopic data showed increased number and decreased size of the mitochondria in a coronary artery ligation rat HF model. Mitochondrial fusion/fission has been relatively recently described, and has not been previously studied in HF; however, mitochondrial morphologic changes have been found in other types of cardiomyopathy. Copper deficiency induced hypertrophy was detected with a dramatic increase in mitochondria, possibly as a compensatory mechanism for a reduction in cytochrome c oxidase and ATP synthase.¹³⁵ With cuprizone, a copper chelator that inhibits monoamine oxidase, this increase is characterized by the appearance of numerous partitioned mitochondria, suggestive of decreased fusion.¹³⁶ Further work will be needed to define the role of abnormal mitochondrial fission and fusion in HF.

Mitochondrial dysfunction has also been reported to lead to upregulation of autophagy, which has been well reviewed by Gustafsson et al.⁸⁶ Autophagy is a process important in cellular homeostasis and in removing excess or damaged organelles.^{137, 138} Overexpression of Bnip3 in HL-1 myocytes was associated both with mitochondrial fragmentation and with increased autophagy.¹³⁹ Autophagy plays an important role in the cellular response to stress and has been shown to be upregulated in the myocardium in response to I/R.^{140, 141} Upregulation of autophagy in HL-1 myocytes was found to protect against simulated I/R cell death in HL-1 myocytes,¹⁴¹ while enhanced autophagy during chronic myocardial ischemia correlated with decreased apoptosis, suggesting that induction of autophagy is a protective response.¹⁴² In contrast, Beclin-1 is a protein essential in the autophagic pathway and heterozygous knockout mice had reduced autophagy, smaller infarcts, and apoptosis after I/R compared to wild type,¹⁴⁰ suggesting that upregulation of autophagy in response to I/R may be detrimental. Clearly, further studies are required to elucidate the roles of mitochondrial fragmentation and autophagy in myocardial cell death and HF.

Despite of the intensive studies of mitochondrial dynamics in other fields, much remains to be understood in the heart. Multiple studies have been done on mitochondrial dynamics in neurologic tissue because of the association of mutations in fusion proteins with inherited optic disorders associated with peripheral neuropathies, such as Charcot-Marie Tooth disease. Further investigations are needed to understand the role of these important proteins involved in fission and fusion in cardiac disease.

Mitochondrial DNA

In addition to the role of mitochondria as a source of ROS, reviewed above, the mitochondria themselves can also be damaged by ROS. ROS may induce mitochondrial DNA (mtDNA) damage, which leads to defects of mtDNA-encoded gene expression and respiratory chain complex enzymes and thus may contribute to the progression of left ventricular (LV) remodeling and failure after myocardial infarction (MI). After MI, a 30–50% decrease in the mtDNA-encoded gene transcripts, as well as the enzymatic activity, including the subunits of complex I, complex III, complex IV, occurred.¹⁴³ In contrast, other studies showed that CHF induced a decrease in oxidative capacity and mitochondrial enzyme activities with a parallel decrease in the mRNA level of COX I and IV, but no change in mtDNA content.¹⁶ However, even with maintenance of mtDNA content, mutation as a result of exposure to increased ROS, can lead to persistent and progressive dysfunction.

Mitochondrial genetic mutations, both mtDNA deletions and tRNA point mutations, can contribute to HF. Mitochondrial genomic changes have not been considered as a cause of cardiac disease, but they are well established as a cause of skeletal muscle myopathy. A clinical study showed that pathological mtDNA mutations are associated with ultrastructurally abnormal mitochondria, defined as giant organelles with abnormal cristae and inclusion bodies, and reduced Cox activity in a small subgroup of idiopathic DCMs, in which mtDNA defects may constitute the basis for, or contribute to, the development of congestive HF.¹⁴⁴ Earlier clinical studies also suggested a relation between mitochondrial mutations and cardiac myopathies. Mitochondrial tRNA(Lys) gene (G8363A) mutation was associated with maternally inherited cardiomyopathy and hearing loss.¹⁴⁵ Further investigation into the contribution of mtDNA changes to HF, especially idiopathic nonischemic cardiomyopathy, is needed.

Aging, mitochondria, and heart failure

The prevalence of HF increases with age. Cardiac myocytes, like other long-lived post-mitotic cells, change significantly with age, compared to the minor to undetectable alterations in short-lived cells, such as intestinal epithelium and bone marrow.^{146, 147} Aging affects cardiac mitochondria at different levels, including increased ROS formation, mutations in mtDNA and mitochondrial fusion/fission balance. Mitochondrial changes related to aging may contribute to the development of HF.

Significant changes in cardiac ultrastructure were found in aged (27 mo) mice. There were giant mitochondria, which had degenerative changes including the disruption, disorganization and loss of cristae and the development of a very electron-lucent matrix.¹⁴⁸ Studies of Syrian hamster LV demonstrated that the area of mitochondrial inner membrane plus cristae per mitochondrial volume decreased in aging, thus there were less cristae per mitochondrion.¹⁴⁹ The formation of giant mitochondria with aging, as found in the mouse and other models ^{148, 150–152} may result from impaired fission/fusion balance, a possibility supported by depressed DNA synthesis in large mitochondria. Navratil et al. found that giant mitochondria accumulated in cultured rat myoblasts due to inhibition of autophagy. These giant mitochondria have low inner membrane potential and do not fuse with each other or with normal mitochondria. Significantly OPA1 was reduced in these mitochondria, but the expression of mitofusin-2 (Mfn2) remained unchanged.¹⁵³ In vitro studies also showed depletion of hFis1, a critical component of mitochondrial fission, resulted in a significant increase of senescence-associated β-galactosidase activity.¹⁵⁴ Thus, giant mitochondria in aging are associated with evidence of decreased fission/fusion, which is known to be important for maintenance of normal mitochondrial function, and with evidence of cell senescence. Both of these changes will lead to significant impairment of myocyte function, and impaired fission/fusion can lead to myocyte loss through apoptosis.

Cardiac mitochondrial mutations have the potential to play an important role in human myocardial aging. Human cardiomyocytes from 350 different donors were scanned for all possible large deletions in mtDNA, deletion-rich cells were found only in the hearts of aged donors, where they occurred at a frequency of up to one in seven cells.¹⁵⁵ Mitochondrial DNA mutations may increase the likelihood of ROS production and further mtDNA lesions.³² Aging cardiac myocytes are subjected to enhanced oxidative stress, which damages mitochondria. Increased mitochondrial ROS formation and oxidative mtDNA damage were detected in aged C57/B16 WT mice, while even higher levels were found in aged MnSOD(+/−) mice.¹⁵⁶ The amount of mitochondrial hydrogen peroxide was also found to increase with age.^{157, 158} Mice overexpressing mitochondrially targeted catalase, an antioxidant, have extended life spans,¹⁵⁹ and have attenuated echocardiographic cardiac aging risk scores, highlighting the critical role of ROS in cardiac aging progress.¹⁶⁰

Estrogen, mitochondria and heart failure

A number of studies suggest that estrogen may affect the cardiac mitochondria directly. Functional estrogen receptors (ERs) (α and β) have been demonstrated in ventricular myocardium of both males and fe-males.^{161, 162} In addition to their presence in the nuclei and plasma membrane, ERα and ERβ are also localized in mitochondria of a number of cell types and tissues. Using subcellular fractionation, immunochemical localization with confocal fluorescent microscopy and immuno EM, Chen et al. ^{163, 164} demonstrated that 20% of cellular ERβ is localized to the mitochondrial matrix of human breast cancer MCF-7, and that 17β-estradiol (E2) enhanced the import of ERβ into MCF-7 cell mitochondria in a time- and dose-dependent manner. Yang et al.¹⁶⁵ demonstrated the mitochondrial localization of ERβ, in primary neurons, primary cardiomyocytes, murine hippocampus cell lines ad human heart cells. The presence of ERα and ERβ in mitochondria of breast, heart, brain, bone, eyes, sperm, and periodontal ligament tissues, which have high energy needs for their function, suggests that mitochondrial ERα and ERβ may have important roles in the regulation of mitochondrial energy metabolism in these systems.

Numerous clinical studies have shown that women have a reduced risk of HF and a better prognosis, should HF develop. An analysis based on the Flolan International Randomized Survival Trial (FIRST) study which enrolled 471 HF patients (359 men) showed a significant association between female gender and better survival (relative risk of death for men versus women was 2.18, 95% CI 1.39 to 3.41; P<0.001).¹⁶⁶ O’Meara et al. compared outcomes in 2 400 women and 5 199 men randomized in the Candesartan in HF: Assessment of Reduction in Mortality and morbidity (CHARM) program using analysis multivariable regression. Female patients had decreased morbidity and mortality, and overall a better prognosis.¹⁶⁷ Another clinical study showed that HF resulted in a 13-fold and 27-fold increase in necrosis in women and men respectively, while apoptosis increased 35-fold in women and 85-fold in men respectively, suggesting the female heart is protected, at least in part, from necrotic and apoptotic death signals.¹⁶⁸ However, other studies suggest different gender effects. In idiopathic dilated cardiomyopathy, females were found to have a significantly poorer prognosis than males.¹⁶⁹ Women may also be more sensitive to alcohol-induced cardiac disease.¹⁷⁰ Thus, a number of studies support a differential gender response to HF, though some controversy remains. Animal studies showed that both the E2/ERα- ^{171, 172} and E2/Eβ,-pathways^{173, 174} were involved in protection against myocardial ischemia-reperfusion injury probably via their effects on the preservation and maintenance of mitochondrial structure. However, the role(s) of estrogens in the cardiac system is still controversial, and the underlying mechanisms unclear.¹⁷⁵ Further studies are needed. Some interesting recent studies support an important role for estrogen in regulation of the expression of mitochondrial genes, as discussed below.

There is increasing evidence that mtDNA is one of the major targets for the direct actions of steroid hormones and their respective receptors.^{162, 176, 177} As recently reviewed by Chen et al., E2 up-regulates the transcript levels of several mtDNA genes, which encode ETC proteins, and has direct and indirect effects on ETC activity.¹⁷⁸ Microarray analysis using ERα and ERβ knockout mice showed that E2/ERβ pathways mediate down-regulation of genes for nuclear DNA-encoded subunits of all ETC, whereas ERα is essential for most of the estrogen-mediated increase in gene expression including ETC proteins and proteins involved in the anti-oxidative stress response.¹⁷⁹ A number of studies investigated E2 and the transcription factors, mostly nuclear factors, regulating genes whose products localize to the mitochondria. Nuclear respiratory factors (NRF)-1 and NRF-2 are the primary transcription factors of nuclear-encoded mitochondrial proteins. Another key transcription factor in the mitochondria is mitochondrial transcription factor A (Tfam), which is a nuclear-encoded, mitochondrially-localized protein that is critical for replication, transcription and maintenance of mtDNA.^{180, 181} Human Tfam binds to mtDNA in a sequence-independent manner and is abundant enough to cover the entire mtDNA genome, thereby stabilizing mtDNA through formation of a nucleoid (mitochromosome, corresponding to chromosome of nuclear DNA) and regulates the amount of mtDNA.¹⁸¹ Several recent studies demonstrated an important role for NRF1 and NRF2 in mediating E2 effects on ETC protein expression.^182–184 Similarly, in rat cerebral blood vessel preparations (composed predominantly of vascular endothelial cells), estrogen treatment enhanced protein levels of cytochrome c, Cox4, as well as NRF1.¹⁸⁴ Furthermore, Eβ· knockout mice showed diminished expression of NRF1¹⁷⁹ Mattingly et al. reported that E2 increased NRF1 mRNA and protein by binding to either ER alpha or ER beta, which then bind an estrogen response element (ERE) in NRF1. The E(2)-induced increase in NRF1 was followed by increased Tfam, the transcription of Tfam-regulated mitochondrial DNA-encoded COXI genes, and increased mitochondrial biogenesis.¹⁸³ Over-expression of human Tfam in mice increased the amounts of mtDNA and dramatically ameliorated cardiac dysfunction post-MI.¹⁸⁵ In contrast, a tissue-specific knock out of Tfam in the mouse heart resulted in dramatic reduction in mtDNA content and impaired mitochondrial function.¹⁸⁶ Knock down of NRF-1 with siRNA blocked E2 stimulation of mitochondrial biogenesis and activity, indicating a mechanism by which estrogen regulates mitochondrial function through increased NRF-1 expression.¹⁸³ Moreover, it has been well known that the transcriptional activities of ERα depend on a co-activator PGC-1, which controls mitochondrial biogenesis and ATP synthesis.^{187, 188} Hsieh et al. have reported ^{182, 189} that E2 increased the expression of rat cardiac PGC-1, NRF-2, Tfam and that these effects were associated with an increase in COX IV and mtDNA-encoded COXI and ATP synthase β-subunit, mitochondrial ATP, and COX activity in rats that underwent trauma-hemorrhage. These effects were totally abolished by the ER antagonist ICI-182780, indicating the involvement of ER in mediating these effects. In a microarray study of failing (32 ischemic cardiomyopathy, 27 idiopathic dilated cardiomyopathy) and control (33) explanted human hearts, Shihag et al.¹⁹⁰ found that a subset of downstream gene targets of the master mitochondrial transcriptional regulator, PGC-1α as well as regulatory partner ERα, decreased in HF. Thus, studies suggest that estrogen can have profound effects on the mitochondria, and much remains to be understood in this area.

Conclusions

HF is a growing clinical problem, in part the result of successes in treating coronary disease. Despite the impact of HF, much remains to be understood about the pathogenesis of this important disease. The development of HF is associated with alterations in a wide variety of pathways, but mitochondria remain at the epicenter of the pathophysiology of this disease. Better comprehension of the mitochondrial changes in HF can potentially lead to the development of new therapeutic strategies.