Targeting mitochondria for cardiovascular disorders: therapeutic potential and obstacles
靶向线粒体治疗心血管疾病：治疗潜力和障碍

Regulated cell death (RCD). A form of cell death that relies on the activation of a genetically encoded machinery and which, therefore, can be retarded or accelerated with specific pharmacological or genetic interventions.
调节细胞死亡 （RCD）。一种细胞死亡形式，依赖于基因编码机制的激活，因此可以通过特定的药物或遗传干预来延缓或加速。

Evolutionarily conserved 进化上守恒
cellular process that culminates with the lysosomal degradation of ectopic, supernumerary, dysfunctional, or potentially dangerous cytoplasmic entities (of endogenous or exogenous derivation).
细胞过程，以异位、多余、功能失调或潜在危险的细胞质实体（内源性或外源性衍生物）的溶酶体降解而告终。

Mitochondria occupy a central position in the biology of most eukaryotic cells, including all the cells of the cardiovascular system, because mitochondria have a major role in catabolic and anabolic metabolism, regulation of intracellular homeostasis, initiation of inflammatory reactions, and control of multiple pathways culminating in regulated cell death (RCD) . In line with this notion, the mitochondrial network is constantly subjected to a tight quality-control system that segregates dysfunctional mitochondria and delivers them to lysosomes for degradation . Such a mechanism, commonly known as mitophagy, involves not only the core molecular machinery for autophagy but also a set of dedicated proteins that are required for the optimal recognition of damaged mitochondria .
线粒体在大多数真核细胞（包括心血管系统的所有细胞）的生物学中占据中心地位，因为线粒体在分解代谢和合成代谢、细胞内 稳态的调节、炎症反应的启动以及最终导致调节细胞死亡 （RCD ） 的多种途径的控制中起主要作用.根据这一概念，线粒体网络不断受到严格的质量控制系统的影响，该系统分离功能失调的线粒体并将它们输送到溶酶体进行降解 。这种机制，通常称为线粒体自噬，不仅涉及自噬的核心分子机制， 还涉及一组最佳识别受损线粒体所需的专用蛋白质 。

A tight control on mitochondrial fitness is paramount for the preservation of cardiovascular homeostasis for at least four reasons . First, cardiomyocytes heavily rely on fatty acid-driven oxidative phosphorylation for ATP production, at least in physiological settings . Thus, a decrease in the bioenergetic efficiency of the mitochondrial network can have a direct detrimental effect on the contractile capacity of cardiomyocytes. Second, fluxes are at the core of overall cardiac activity . Therefore, defects in the capacity of the mitochondrial network (in conjunction with the endoplasmic reticulum) to regulate homeostasis can alter cardiac functions such as electrical conduction. Third, physiological inflammatory homeostasis is particularly important not only for normal cardiac functions but also for the preservation of vascular compartments . Thus, damaged mitochondria accumulating in the cytosol of cardiomyocytes or endothelial cells can drive pathogenic inflammatory responses. Finally, the integrity of the cardiovascular system is crucial for optimal contractile and circulatory functions . Severe mitochondrial dysfunction and/or the accumulation of permeabilized mitochondria (beyond a threshold that depends on multiple parameters) can initiate several variants of RCD that culminate in pathological tissue loss (FIG. 1).
严格控制线粒体适应性对于保持心血管稳态至关重要，原因至少有四个 。首先，心肌细胞严重依赖脂肪酸驱动的氧化磷酸化来产生 ATP，至少在生理环境中 是这样。因此，线粒体网络生物能量效率的降低会对心肌细胞的收缩能力产生直接的不利影响。其次， 通量是整体心脏活动 的核心。因此，线粒体网络（与内质网结合）调节 体内平衡的能力缺陷可以改变心脏功能，例如导电。第三，生理炎症稳态不仅对正常的心脏功能 尤为重要，而且对血管隔室的保存也尤为重要 。因此，受损的线粒体在心肌细胞或内皮细胞的胞质溶胶中积累可以驱动致病性炎症反应。最后，心血管系统的完整性对于最佳收缩和循环功能 至关重要。严重的线粒体功能障碍和/或透化线粒体的积累（超过取决于多个参数的阈值）可引发 RCD 的几种变体，最终导致病理组织丢失（图 1）。

In line with these observations, mitochondrial defects have been involved, at least to some extent, in the pathogenesis of a variety of cardiovascular disorders, including (but not limited to) myocardial infarction (MI), cardiomyopathies of different aetiology, some forms of arrhythmia, hypertension, atherosclerosis, and other vascular conditions . Starting in the late 1990s, the identification of mitochondrial dysfunction as a central aetiological determinant of cardiovascular disease (CVD) drove an intensive wave of preclinical and clinical investigation aimed at the development of novel targeted therapies . Thus far, the results of such an effort have been disappointing, as no molecules specifically conceived to target mitochondria are currently available for use against CVD in clinical settings . In this Review, we discuss the rationale for using mitochondriatargeting agents (MTAs) in the treatment of CVD, dissect the obstacles that have limited their development over the past 2 decades, and put forward strategies that might unleash the full potential of these promising - but hitherto unrealized - therapeutic tools.
根据这些观察结果，线粒体缺陷至少在某种程度上参与了多种心血管疾病的发病机制，包括（但不限于）心肌梗死 （MI）、不同病因的心肌病、某些形式的心律失常、高血压、动脉粥样硬化和其他血管疾病 .从 1990 年代后期开始，线粒体功能障碍被确定为心血管疾病 （CVD） 的核心病因决定因素，推动了一波密集的临床前和临床研究浪潮，旨在开发新的靶向疗法 。到目前为止，这种努力的结果令人失望，因为目前没有专门针对线粒体的分子可用于在临床环境中 对抗 CVD。在这篇综述中，我们讨论了使用线粒体靶向剂 （MTA） 治疗 CVD 的基本原理，剖析了过去 2 年中限制其发展的障碍，并提出了可能释放这些有前途但迄今为止尚未实现的治疗工具的全部潜力的策略。

Biochemical pathway whereby
生化途径
fatty acids are converted into
脂肪酸转化为
acetyl-CoA, which enters the
乙酰辅酶 A，它进入
TCA cycle, and NADH and
TCA 循环以及 NADH 和
FADH 2 , which fuel oxidative phosphorylation. 

Biochemical pathway whereby 
ketone bodies are converted 
into acetyl-CoA, which enters 
the TCA cycle, and NADH, 
which fuels oxidative 
phosphorylation. 

Targeting mitochondria from multiple angles has been associated with beneficial effects in a variety of experimental CVD models (TABLES 1,2). However, limited benefits have been documented in clinical trials investigating the safety and efficacy of MTAs for the treatment of CVD, as discussed below. 
Mitochondrial metabolism. Healthy cardiomyocytes satisfy their elevated energy needs by catabolizing fatty acids (via -oxidation), branched-chain amino acids, and, to a lesser extent, ketone bodies (via ketolysis) to fuel the tricarboxylic acid (TCA) cycle and drive ATP production via the mitochondrial respiratory chain (BOX 1). By comparison, pyruvate derived from glycolysis contributes minimally to ATP synthesis in the healthy heart . Such a predominantly mitochondrial metabolic profile 

shifts in the course of numerous cardiac pathologies. Heart failure (HF) is accompanied by a gradual decline in the bioenergetic reserve capacity of the myocardium, which - beyond a specific threshold - can no longer be compensated for by endogenous mechanisms . In multiple variants of cardiomyopathy culminating with HF , cardiomyocytes undergo metabolic reprogramming involving decreased -oxidation and branched-chain amino acid metabolism coupled with intracellular lipid deposition and increased glucose utilization . The TCA cycle intermediate succinate accumulates in the ischaemic myocardium, and such an accumulation is mechanistically linked to oxidative damage at reperfu (see below). Along similar lines, TCA cycle activity is impaired 6 weeks after , potentially representing an early maladaptive phase of the surviving tissue. 

The molecular mechanisms underlying metabolic reprogramming in the diseased myocardium remain to be fully elucidated, although a role for specific transcription factors has been postulated. For instance, nuclear receptor subfamily 2 , group F, member 2 (NR2F2; also known as COUP-TF2) is upregulated in patients with HF, and transgene-driven Nr2f2 overexpression in mice favours dilated cardiomyopathy accompanied by pathological metabolic remodelling . Similarly, hypoxia-inducible factor (HIF1 ) initiates a transcriptional programme involving peroxisome proliferator-activated receptor- (PPAR ) that leads to increased glucose uptake and consequent lipid accumulation, apoptotic cell death, and contractile dysfunction . Corroborating an aetiological role for this transcriptional module, ventricular-specific deletion of Hifla prevents pressure-overload-induced cardiomyopathy in mice . 

Additional metabolic functions ensured (at least in part) by mitochondria are relevant for CVD, including the folate cycle. An efficient folate cycle is indeed required for the optimal conversion of homocysteine into methionine, and defects in this pathway, including genetic variants in MTHFR (which encodes methylenetetrahydrofolate reductase) are associated with an increased incidence of vascular disorders (such as thrombosis and atherosclerosis) secondary to, or at least paralleled by, homocysteine accumulation . Of note, several mutations in mitochondrial or nuclear genes coding for components of the mitochondrial respiratory chain have been associated with familial cardiomyopathies in humans . Moreover, experimental interventions inducing respiratory defects in myocardial cells, such as the tissue-specific deletion of Aifm1 (which encodes apoptosis inducing factor mitochondria associated 1 or Tfam (which encodes mitochondrial transcription factor A; TFAM , result in spontaneous, early-onset cardiomyopathy. Taken together, these observations exemplify the involvement of mitochondrial metabolic dysfunction in CVD. 

Early clinical trials testing L-carnitine supplementation, which (among other effects) favours the mitochondrial uptake of cytosolic fatty acids, in patients recovering from acute MI documented some degree of efficacy in reducing the incidence or severity of HF, left ventricular enlargement, arrhythmias, and cardiac death . However, subsequent studies did not 

Fig. 1 | Contribution of mitochondrial dysfunction to cardiovascular disease. In physiological conditions, healthy mitochondria support the functions of virtually all cells from the cardiovascular system by ensuring optimal catabolic and anabolic metabolism and regulating the intracellular trafficking of . Additionally, an intact mitochondrial network promotes the preservation of inflammatory homeostasis and tissue integrity by preventing the activation of signal transduction cascades that lead to the release of pro-inflammatory factors and regulated cell death. In addition to being accompanied by metabolic derangements and alterations in intracellular fluxes, mitochondrial dysfunction favours the establishment of an inflammatory milieu and facilitates regulated cell death, which culminates with tissue loss. By efficiently eliminating dysfunctional mitochondria that originate as a consequence of physiological cellular functions or accumulate in the context of pathological cues, mitophagy has a major role in the preservation of cardiovascular homeostasis. 

Biochemical pathway 
catalysing the cyclic conversion of tetrahydrofolate, 10 -formyltetrahydrofolate (which feeds 
into purine synthesis), 
5,10-methylenetetra- 
hydrofolate, and 5-methyl- 
tetrahydrofolate (which feeds 
into methionine metabolism) 
Mitochondrial permeability transition 
(MPT). Rapid loss of the ionic barrier function of the inner mitochondrial membrane, culminating in mitochondrial breakdown and regulated necrosis. conclusively confirm these observations . Moreover, oral L -carnitine can be metabolized by the gut microbiota into trimethylamine -oxide (TMAO), a proatherogenic molecule . Accordingly, individuals with high L-carnitine levels and concurrently high TMAO levels in the blood are at increased risk of CVD and major adverse cardiac events . Thus, the clinical development of L-carnitine for the treatment of CVD seems to be at an impasse. 

The -oxidation inhibitor etomoxir has also been investigated in patients with congestive HF, with inconclusive results . Conversely, perhexiline and trimetazidine - which resemble etomoxir in their capacity to inhibit -oxidation (although to different degrees) are currently approved in multiple countries (including Australia and Canada) as antianginal agents . The therapeutic efficacy of perhexiline and trimetazidine has been proposed not to reflect a switch from fatty acid-driven to glucose-driven catabolism but instead to entail an entire rebalancing of carbon and nucleotide phosphate fluxes linked to autophagy activation (see below). 
Perhexiline is also effective (at least to some extent) in a subset of patients with cardiomyopathy , but not in patients with left ventricular hypertrophy undergoing cardiac surgery . Trimetazidine has been tested in multiple cohorts of patients with distinct cardiovascular disorders beyond angina, with variable degrees of efficacy . Nonetheless, in the USA (but not in other countries), the clinical development of perhexiline and trimetazidine has been discontinued, presumably owing to a fairly narrow therapeutic index . 
5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR; also known as acadesine) is an intermediate in the synthesis of inosine monophosphate that potently activates -AMP-activated protein kinase (AMPK), a metabolic sensor regulating mitochondrial biogenesis, dynamics, and metabolism . Despite some promising preliminary results , the clinical development of acadesine as a cardioprotective intervention in patients undergoing CABG surgery has been abandoned, at least in part owing to the lack of long-term efficacy . In summary, despite a robust rationale to target mitochondrial metabolism for the prevention or treatment of CVD, this therapeutic strategy remains largely unrealized. 

Sirtuins. Sirtuins are a family of -dependent deacetylases and deacylases that control multiple aspects of cellular metabolism, including mitochondrial function and redox balance . The mammalian genome encodes seven different sirtuins, three of which (SIRT3, SIRT4, and SIRT5) are localized to mitochondria . Pharmacological sirtuin activation mediates lifespan-extending functions in multiple experimental models , and defects in both mitochondrial and extramitochondrial sirtuins have been associated with a variety of cardiovascular disorders . Sirt1 mice are viable but have considerable developmental heart defects . In Sirt1 hearts, ischaemic preconditioning does not preserve cardiac function after ischaemiareperfusion injury, potentially linked to hyperacetylation of cytosolic proteins and consequent inhibition of autophagy , whereas myocardial Sirt1 overexpression has cardioprotective effects along with deacetylation of cytoplasmic proteins . Sirt3 mice show signs of cardiac hypertrophy and interstitial fibrosis at 8 weeks of age, spontaneously develop age-related cardiomyopathy, and are more sensitive than their wild-type littermates to hypertrophic stimuli, including aortic constriction . Such a susceptibility to cardiac hypertrophy reflects, at least in part, an increased propensity of the Sirt3 myocardium to undergo regulated necrosis upon mitochondrial permeability transition (MPT) as a consequence of cyclophilin D (CypD; also known as PPIF) hyperacetylation (see below). Conversely, transgenic Sirt 3 overexpression has robust cardioprotective effects in mice . Similar results to those observed in Sirt3 mice have been obtained with Sirt2 , Sirt5 , Sirt6 , and Sirt7-/- mice, and as shown with Sirt3 overexpression, overexpression of Sirt2 specifically in the myocardium had cardioprotective effects . By contrast, Sirt4 mice seem to be less susceptible to angiotensin-II-induced cardiac hypertrophy than their wild-type counterparts, whereas cardiomyocyte-specific
Sirtuins 的。Sirtuins 是一个 依赖性脱乙酰酶和脱酰酶家族，控制细胞代谢的多个方面，包括线粒体功能和氧化还原平衡 。哺乳动物基因组编码 7 种不同的 sirtuin，其中 3 种（SIRT3、SIRT4 和 SIRT5）定位于线粒体 。药理学 sirtuin 激活在多个实验模型中 介导延长寿命的功能 ，线粒体和线粒体外 sirtuin 的缺陷都与多种心血管疾病 有关。Sirt1 小鼠是可行的，但有相当大的发育性心脏缺陷 。在 Sirt1 心脏中，缺血预处理在缺血再灌注损伤后不能保持心脏功能，可能与胞质蛋白的高乙酰化和随之而来的自噬抑制有关 ，而心肌 Sirt1 过表达与细胞质蛋白 的脱乙酰化一起具有心脏保护作用。Sirt3 小鼠在 8 周龄时表现出心脏肥大和间质纤维化的迹象，自发发展为与年龄相关的心肌病，并且比其野生型同窝小鼠对肥大刺激（包括主动脉收缩 ）更敏感。这种对心脏肥大的易感性至少部分反映了由于亲环蛋白 D （CypD;也称为 PPIF） 高乙酰化 （见下文），Sirt3 心肌在线粒体通透性转换 （MPT） 时发生调节坏死的倾向增加。相反，转基因 Sirt 3 过表达在小鼠 中具有强大的心脏保护作用。 Sirt2 、 Sirt5 、 Sirt6 和 Sirt7 - /- 小鼠获得了与在 Sirt3 小鼠中观察到的结果相似的结果，并且如 Sirt3 过表达所示，Sirt2 特异性在心肌中的过表达具有心脏保护作用 。相比之下，Sirt4 小鼠似乎比野生型小鼠更不易患血管紧张素 II 诱导的心脏肥大，而心肌细胞特异性

Table 1 | Genetic studies implicating mitochondrial functions in cardiovascular physiology in mice
表 1 |线粒体功能与小鼠心血管生理学有关的遗传研究

DNM1L, dynamin-1-like protein; MFN2, mitofusin 2; PARK2, parkin RBR E3 ubiquitin protein ligase.
overexpression of Sirt4 reportedly mediates detrimental effects in this model . However, these findings have not yet been confirmed. At least in part, the cardioprotective effects of sirtuin activation originate from an antioxidant transcriptional programme orchestrated by forkhead box protein O3A (FOXO3A; also known as FOXO3) , proficient autophagic responses , and potentially the inhibition of MPT-driven regulated necrosis (see below). Thus, sirtuins support cardiac fitness by affecting mitochondrial functions.

Sirtuins are activated by caloric restriction, which is also a potent inducer of autophagy, and a vast amount of literature is available on the multipronged beneficial effects of caloric restriction on cardiovascular health in humans, at least part of which are thought to depend mechanistically on sirtuins . Additional sirtuin activators include the rather nonspecific natural polyphenols butein, honokiol, piceatannol, quercetin, and resveratrol as well as several synthetic sirtuin-activating compounds, including SRT1720, SRT2104, and SRT3025 (REF. ). All these molecules have been shown to mediate beneficial effects in rodent models of CVD, and both SRT1720 and SRT2104 extend mouse lifespan . Similarly, dietary supplementation with nicotinamide mononucleotide ( NMN ; a precursor of ) mediates potent cardioprotective effects in mouse models of cardiomyopathy and ischaemia-reperfusion injury via a SIRT1-dependent or SIRT3-dependent mechanism . The capacity of dietary resveratrol to limit the incidence or severity of various cardiovascular disorders (mostly in the context of type 2 diabetes mellitus) has been investigated in multiple clinical trials , with inconclusive findings (often due to problematic study design). Still, no fewer than 20 non-closed (status: not terminated, suspended, or withdrawn) clinical trials are currently registered at clinicaltrials.gov to investigate dietary supplementation with resveratrol in individuals with age-associated morbidities (mostly type 2 diabetes) and cardiovascular conditions including non-ischaemic cardiomyopathy (NCT01914081), hypertension (NCT01842399), atherosclerosis (NCT02998918), and endothelial dysfunction (NCT02256540). Results from a small randomized clinical trial including 40 patients with psoriasis (NCT01154101) suggest that SRT2104 is well tolerated . The safety of SRT3025 has been investigated in healthy volunteers (NCT01340911), but to the best of our knowledge the results of this study have not been disseminated. Finally, the effects of dietary NMN supplementation on cardiometabolic functions are currently being formally investigated (NCT03151239). Taken together, these observations suggest that, although multiple dietary interventions that activate sirtuins, including caloric restriction, resveratrol, and NMN (both of which are available over the counter), might mediate robust cardioprotective effects, additional clinical testing is required for the establishment of official treatment protocols enabling the use of these agents for the treatment of CVD.

Mitochondrial dynamics. The mitochondrial network constantly undergoes remodelling owing to the mutually antagonistic activity of multiple proteins that promote fission, such as mitochondrial fission factor (MFF), mitochondrial fission 1 protein (FIS1), and dynamin-1like protein (DNM1L), and fusion, such as mitofusin 1 (MFN1), MFN2, and optic atrophy protein 1 (OPA1) (FIG. 2). This process is paramount for the preservation of optimal mitochondrial functions in both physiological and pathological conditions, at least in part because fission enables the mitophagic disposal of dysfunctional mitochondria . Accordingly, multiple genetic defects impairing mitochondrial dynamics have been linked to CVD in experimental models.

The myocardium of mice has clustered mitochondria with disorganized cristae and reduced mitochondrial DNA (mtDNA) content, and Opal mice are more susceptible to cardiac hypertrophy induced by transverse aortic constriction than their wild-type counterparts . Cardiomyocyte-specific deletion of Yme1l1 accelerates cardiac OPA1 proteolysis, thereby favouring mitochondrial hyperfragmentation and metabolic impairment, leading to . Interestingly, angiotensin-II-induced cardiomyopathy leads to OPA1 acetylation and consequent mitochondrial fragmentation, a detrimental process that is inhibited by SIRT3 (REF. ). The co-deletion of and from adult cardiomyocytes imposes a robust defect in mitochondrial fusion that drives cardiac dysfunction associated with rapidly progressive (and ultimately lethal) dilated cardiomyopathy . Such a detrimental phenotype cannot be fully rescued by the concomitant deletion of Dnm1l, but the cardiomyopathy manifesting in hearts progresses with different kinetics than in hearts and mostly reflects a mitophagic blockage . However, hearts have reduced sensitivity to ischaemia-reperfusion injury compared with their wild-type counterparts, potentially as a consequence of mitigated overload (see below).

Transgenic expression of DNM1L-C452F (a hyperactive DNM1L variant) also drives dilated cardiomyopathy accompanied by a considerable mitophagic defect . Similarly, mouse hearts spontaneously develop dilated cardiomyopathy accompanied by mitochondrial hyperfragmentation, impaired contractile performance, and insensitivity to -adrenergic stimulation . Further corroborating the importance of mitochondrial fusion for the preservation of cardiovascular homeostasis, adenovirus-mediated delivery of to the mouse myocardium inhibits angiotensin-IIinduced cardiomyopathy . Interestingly, transgenedriven overexpression of a non-phosphorylatable MFN2 variant (MFN2-AA) in the myocardium of newborn (but not adult) mice prevents normal mitochondrial maturation, accompanied by a switch from glucose-driven to fatty acid-driven metabolism, and leads to premature lethality, most probably as a consequence of impaired mitophagy (see below). Of note, physiological DNM1L-dependent mitochondrial fragmentation is critical for cardiac adaptation to increased energy demands . Moreover, conditional deletion of one copy of Dnm1l from the myocardium exacerbates pressure-overload-induced cardiomyopathy as well as ischaemia-reperfusion injury in mice as a consequence of mitophagy impairment . Altogether, these observations suggest that a balanced interplay between fission

Table 2 | Genetic studies implicating mitochondrial functions in cardiovascular pathology in mice

Table 2 (cont.) | Genetic studies implicating mitochondrial functions in cardiovascular pathology in mice

, mitochondrial transmembrane potential; CaMKII, calcium/calmodulin-dependent protein kinase II; DN, dominant-negative; HFD, high-fat diet; IPC, ischaemic preconditioning; I/R, ischaemia-reperfusion; MCU, calcium uniporter protein, mitochondrial; MPT, mitochondrial permeability transition; NF- KB , nuclear factor- kB ; RCD, regulated cell death; ROS, reactive oxygen species; SIRT3, sirtuin 3.
and fusion is paramount for cardiovascular health as it preserves mitochondrial fitness in both physiological and pathological conditions. Further corroborating this notion, the levels of various factors involved in the regulation of mitochondrial dynamics, including FIS1, MFN2, and OPA1, are altered in the course of CVD . Of note, MFN2 is also aetiologically involved in the proliferative arrest and death of vascular smooth muscle cells elicited by oxidative stress in rats . In line with this notion, transgene-driven Mfn2 overexpression reportedly prevents vascular smooth muscle cell proliferation and restenosis in rat models of

Oxidative phosphorylation is a core bioenergetic process whereby reducing equivalents present in the mitochondrial matrix are sequentially used by four multiprotein complexes (generally referred to as respiratory complexes I-IV) and two electron shuttles (namely, coenzyme and cytochrome ) to generate an electrochemical gradient across the inner mitochondrial membrane that is harnessed in a controlled manner by the ATP synthase (also known as respiratory complex V ) to catalyse the phosphorylation of ADP into ATP. The main substrates for oxidative phosphorylation are NADH, which provides electrons to complex I (also known as NADH dehydrogenase), and succinate, which provides electrons to complex II (also known as succinate dehydrogenase) via . Accordingly, can also fuel oxidative phosphorylation at the level of complex II. Both complex I and II deliver electrons to complex III (also known as CoQ:Cyt c oxidoreductase) via CoQ. However, only complex I transfers electrons onto complex III while also extruding ions from the mitochondrial matrix to the intermembrane space. Complex III transfers electrons to complex IV (also known as Cyt c oxidase) via Cyt c, culminating with the reduction of into . This last step is the reason why is critical for oxidative phosphorylation. Both complex III and complex IV directly contribute to the generation of the mitochondrial transmembrane potential . Finally, the ATP synthase uses a well-described rotatory mechanism to dissipate the in a controlled manner, coupled with phosphorylation of ADP into ATP. This reaction requires ADP and inorganic phosphate ( ), which are provided by the permeability transition pore components adenine nucleotide translocator (ANT) and phosphate carrier (PHC; also known as SLC25A3), respectively (see the figure; please note that stoichiometry is not respected for the sake of simplification). Importantly, the reaction catalysed by the ATP synthase is reversible. This reversibility implies that in ischaemic conditions the capacity of oxidative phosphorylation to drive ATP synthesis is impaired, owing to limited oxygen availability, and that high amounts of ATP are consumed by the ATP synthase to preserve the . All metabolic intermediates entering the tricarboxylic acid (TCA) cycle, including (but not limited to) glucose-derived pyruvate and branchedchain amino acid-derived and fatty acid-derived acetyl-CoA and succinyl-CoA, can drive the synthesis of NADH and succinate in the mitochondrial matrix, thereby supporting oxidative phosphorylation. Fatty acid oxidation also supports oxidative phosphorylation via synthesis. Of note, the cellular efficiency of oxidative phosphorylation depends on a variety of parameters, including the number of mitochondria per cell and their fragmentation state, the amount of respiratory complexes per mitochondrion, the supramolecular organization of respiratory complexes, substrate and availability, the expression of endogenous inhibitors, and local redox and pH conditions .

Intermembrane space

arterial injury induced by balloon denudation of the left common carotid artery . However, these effects seem to be independent of the role of MFN2 in the regulation of mitochondrial dynamics .

The chemical DNM1L inhibitor mdivi-1 mediates cardioprotective effects in rodent models of cardiac ischaemia-reperfusion injury and cardiomyopathy , but the specificity of mdivi-1 has been questioned . Nonetheless, similar observations have been made with other DNM1L inhibitors such as P110 (REFS ) and dynasore . A cell-permeant peptide enabling MFN2-dependent mitochondrial fusion has also been developed , but its biological activity in the cardiovascular system remains to be investigated. To the best of our knowledge, none of these agents has been tested in clinical settings thus far.

Mitophagy. Mitophagy constitutes a pillar in the maintenance of mitochondrial homeostasis in both the healthy and diseased cardiovascular system . Accordingly, multiple defects in the molecular apparatus underlying proficient mitophagic responses have been associated with spontaneous CVD in experimental models . Pink1 mice (lacking a kinase involved in the recognition of depolarized mitochondria) develop left ventricular dysfunction and cardiac hypertrophy by 2 months of age . Deletion of Park2 (also known as Prkn; encoding parkin RBR E3 ubiquitin protein ligase, a functional mitochondrial interactor of serine/threonine protein kinase PINK1, which is required for multiple variants of mitophagy) from the myocardium of adult mice causes a very mild cardiac phenotype in unstressed animals . Conversely, Park2 ablation from the myocardium of neonate mice causes premature and rapidly lethal cardiomyopathy associated with failed mitochondria maturation (strikingly similar to the phenotype associated with MFN2-AA expression) . Similarly, knockout of park (the fly orthologue of Park2) in Drosophila melanogaster causes dilated cardiomyopathy that can be rescued by cardiomyocyte-specific re-expression of park . Bnip mice lack a core component of the molecular apparatus for mitophagy and spontaneously develop cardiomegaly and contractile depression by 60 weeks of age, a pathological phenotype that is further accelerated by the concomitant deletion of Bnip3 (coding for yet another protein involved in mitophagy) . Genetic defects affecting autophagy also compromise cardiovascular homeostasis owing to the accumulation of dysfunctional mitochondria. This observation holds true for: cardiomyocyte-specific deletion of in adult mice, which causes lethal cardiac hypertrophy accompanied by disorganized sarcomere structure as well as mitochondrial misalignment and aggregation ; whole-body deletion of Fbxo32 in mice, which is associated with premature death owing to cardiac degeneration associated with deficient autophagic responses ; and the Lamp2 genotype, which causes a major lysosomal dysfunction that, in mice, drives a vacuolar myopathy that affects cardiac and skeletal muscles, resembling Danon disease . Of note, multiple genetic and pharmacological interventions that impair mitochondrial dynamics impose at least some degree of mitophagic

Fig. 2 | Overview of mitochondrial dynamics. The mitochondrial network is constantly reshaped by the antagonistic activity of proteins that mediate fission, such as mitochondrial fission factor (MFF), mitochondrial fission 1 protein (FIS1), and dynamin 1-like protein (DNM1L), and proteins that promote fusion, such as mitofusin 1 (MFN1), MFN2, and optic atrophy protein 1 (OPA1). One of the essential roles of fission is to segregate dysfunctional mitochondria, thereby enabling their uptake by the autophagic machinery and consequent degradation in lysosomes. PARK2, parkin RBR E3 ubiquitin protein ligase; PINK1, PTEN-induced putative kinase protein 1.

Iron-binding plasma
glycoprotein that controls the level of free iron ions in biological fluids. incompetence . These two processes are so intimately interconnected that mechanistically ascribing the phenotype to either of the alterations is difficult. Additional genetic alterations that trigger CVD in rodents, such as cardiac deletion of Tfrc (coding for the transferrin receptor) , are associated with mitophagic defects. Moreover, genetic defects that improve mitophagic proficiency, such as whole-body absence of (also known as ; coding for a master regulator of cellular biology that inhibits autophagy in physiological settings), decelerate spontaneous cardiac ageing . Taken together, these observations exemplify the critical role of mitophagy in the preservation of physiological cardiovascular homeostasis. That said, Park2 deletion seems to rescue, at least in part, the lethal phenotype of Dnm1l deletion in the adult myocardium , suggesting a role for uncontrolled mitophagy in the detrimental phenotype imposed by defects in mitochondrial fission (see above).
Multiple genetic defects impairing mitophagic proficiency aggravate disease severity in experimental models of CVD . Bnip Bnip hearts are highly sensitive to decompensation induced by pressure overload . Homozygous or heterozygous deletion of Atg5 from the mouse myocardium exacerbates cardiomyopathy driven by pressure overload and angiotensin II administration . Similarly, mice bearing Atg5 monocytes are more susceptible to develop atherosclerotic lesions in response to a high-fat diet or deletion than mice with wild-type monocytes . Mice engineered to overexpress Rheb, which encodes the endogenous autophagy inhibitor RAS homologue enriched in brain (RHEB), in the myocardium are more susceptible to cardiac ischaemia-reperfusion injury than wild-type mice, a detrimental phenotype that can be partially rescued by administration of the pharmacological autophagy activator rapamycin . Dnase mice, which lack a lysosomal nuclease (deoxyribonuclease ) that is involved

Cerebral cavernous malformations
Cerebrovascular disease characterized by enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral haemorrhages.

Member of a fairly new class of targeted anticancer agents that operate by derepressing histone acetylation, resulting in the epigenetic reconfiguration of multiple transcriptional modules. in the autophagic degradation of mtDNA released upon mitochondrial damage, are extremely sensitive to pressure-overload-induced cardiomyopathy, at least in part owing to exaggerated inflammatory responses in the myocardium (see below). Interestingly, cathelicidin antimicrobial peptide (CAMP) can bind mtDNA to limit its degradation by DNase (DNASE2 2 ), which has been associated with exacerbated atherosclerosis in Apoe mice .

Whole-body overexpression of Atg7 (encoding a core component of the autophagic machinery) restrains cardiac hypertrophy and extends survival in a mouse model of desmin-related cardiomyopathy . The genotype limits both ischaemia-reperfusion injury and doxorubicin cardiotoxicity in mice, potentially owing to reduced myocardial susceptibility to RCD (see RCD section below), and improved mitophagy . Multiple other genetic alterations that mediate beneficial effects in experimental models of CVD are associated with superior mitophagic responses (although precise mechanistic links are missing), including the Stk genotype, which limits cardiac ischaemia-reperfusion injury , and the whole-body deletion of Lclat1, which mitigates hypertrophic cardiomyopathy induced by thyroid hyperstimulation . Moreover, multiple cardioprotective interventions including hypothermia and the administration of glucagon-like peptide 1 receptor (GLP1R) agonists have been shown to promote autophagy (at least in some cell types), correlating with reduced amounts of RCD