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Aging and loss of bioenergetic capacity
Aging is a complex biological process associated with a progressive decline in the physiological and biochemical performance of individual tissues and organs, leading to age-associated disease and senescence [1]. Fatigue, weakness, loss of appetite, weight loss and falls are common in the aging population. It has been proposed that a loss of bioenergetic capacity of cells contribute to this progressive loss of function with age [2,3]. The adult human body hydrolyses as much as 64 kg of
Role of mitochondria in cellular bioenergetics
Majority of cellular energy is provided by mitochondria that generate ATP by oxidative phosphorylation. Mitochondria are composed of two membranes, a smooth outer mitochondrial membrane (OMM) and an inner mitochondrial membrane (IMM) composed of numerous invaginations called cristae. In tissues with high energy demand, such as the heart, mitochondria are elongated, and the cristae membranes are closely stacked and take up most of the mitochondrial volume. Embedded in the cristae membranes are
Aging and mitochondrial dysfunction
Aging is associated with alterations in both mitochondrial form and function. Mitochondria become more round in aged cardiomyocytes, with fewer and shorter cristae [[4], [5], [6]]. There is also a significant decrease in number and size of mitochondria in aged human retinal pigment epithelium as well as loss of cristae [7]. The loss of cristae membranes associated with aging impairs the supply of ATP necessary to maintain essential cellular functions. Aging is associated with decreased activity
Improving mitochondrial function to minimize age-associated debilitating conditions
Current approaches toward improving mitochondrial ATP production in aging center on nutritional supplements such as α-lipoic acid and acetyl-L-carnitine, cofactors like nicotinamide adenine dinucleotide (NAD+), and reducing mitochondrial oxidative stress. NAD+ is a central metabolic cofactor that plays a critical role in regulating cellular metabolism and mitochondrial bioenergetics. In its reduced form, NADH serves as the primary electron donor in the ETC. NAD+ is markedly reduced with age,
Targeting cardiolipin to optimize the mitochondrial ETC
Despite the many approaches being pursued for mitochondrial function, none of them directly target the ETC to improve coupling efficiency and increase ATP production. Electron transfer and ATP production require numerous proteins to work in concert on the IMM. Targeting a single protein is unlikely to be successful, and development of mitochondrial therapeutics remain a challenge.
Recent studies have brought attention to cardiolipin as a unique target for promoting mitochondrial efficiency [38,39
Aging and cardiolipin depletion
Cardiolipin content is reduced in the aged heart while there is increase in oxidized cardiolipin [9,15,65,66]. Cardiolipin is highly prone to peroxidation of its acyl chains because of their unsaturated double bonds. Cardiolipin can be oxidized by mitochondrial hydrogen peroxide, but this oxidation is catalyzed by cytochrome c [67]. Hydrophobic binding of cardiolipin to cytochrome c disrupts heme ligation in cytochrome c to confer peroxidase activity [68]. The unleashed peroxidase selectively
Serendipity and the discovery of cardiolipin-targeted peptides
Prevention of cardiolipin loss during aging may help preserve mitochondrial bioenergetics and restore cellular energetics for normal function. Chance discovery led to the identification of a family of small cell-penetrating peptides that selectively target cardiolipin on the IMM [38,39]. The Szeto-Schiller (SS) peptides are synthetic tetrapeptides with an alternating aromatic-cationic motif (Fig. 1). There are a number of surprising features about these peptides. Despite being highly
SS peptides promote oxidative phosphorylation and reduce ROS production
Once the SS peptides bind cardiolipin on the IMM, they are able to promote electron flux in the ETC. Addition of SS peptides to isolated mitochondria increases oxygen consumption and ATP production regardless of whether CI or CII substrates are used [[79], [80], [81]]. Electron transfer from CIII to CIV via cytochrome c is considered the rate-limiting step in the ETC. Electrostatic interaction with cardiolipin is advantageous for keeping cytochrome c close to the respiratory complexes. However,
SS peptides inhibit cardiolipin peroxidation
By protecting the hexacoordination of the cytochrome c heme, these peptides inhibit cytochrome c peroxidase activity and prevent cardiolipin peroxidation [[79], [80], [81]]. In addition, protection of the Met80-Fe ligation prevents self-oxidation of cytochrome c Met80 and exposing the reactive Fe2+ which can oxidize mitochondrial lipids and proteins [80]. Peroxidized cardiolipin can further elicit a free radical chain reaction even without further ROS generation [82]. Quantitation of
SS-31 rapidly rejuvenates oxidative phosphorylation in aged skeletal muscle
Reduced exercise tolerance in aged humans and mice has been associated with impaired bioenergetics and metabolism in aging skeletal muscle [84]. Elderly humans (65–80 years) have significantly lower lean body mass compared to young adults. Muscle biopsies revealed higher oxidative damage as well as lower mitochondrial DNA content. Key oxidative enzyme activity is lower in older subjects and there is a continuous decline in ATP production as a function of age that cannot be accounted for by the
SS-31 remodels mitochondria cristae structure in aged mice
Mitochondria number is considered a major factor for ATP production, and exercise training is believed to increase mitochondria number in skeletal muscle. Interestingly, a recent study revealed that endurance-trained athletes with above normal oxidative capacity have higher cristae density, but not mitochondrial volume, in the leg muscle, compared to recreationally active or sedentary obese individuals [90]. Until recently, it was thought that increasing mitochondria number is the only way to
SS-31 upregulates enzymes required for cardiolipin biosynthesis and remodeling
The ability of SS peptides to repair mitochondrial cristae in aged mice suggests that these compounds can promote cardiolipin biosynthesis. Besides aging, a loss of 18:2 cardiolipin is seen in both experimental and human heart failure [[98], [99], [100]]. Diet-induced metabolic syndrome in pigs induced loss of 18:2 cardiolipin in both heart and kidney [96,101]. The loss of cardiolipin is associated with mitochondrial oxidative stress [99,100,102]. Three months treatment with SS-31 in dogs with
SS-31 restores mitochondrial biogenesis
Mitochondrial biogenesis is known to be impaired in aging and in a variety of diseases [103]. PGC-1α is the major regulator of mitochondrial biogenesis. PGC-1α activates nuclear respiratory factors (NRF-1 and NRF-2) to increase the expression of mitochondrial transcription factor A (TFAM). PCG-1α is down-regulated in skeletal muscle with age, and increased expression of PGC-1α in skeletal muscle improves exercise capacity and prevents muscle wasting in aged mice [104,105]. PGC-1α were reported
SS-31 restores mitochondrial dynamics
Mitochondria are dynamic organelles constantly moving within the cell in response to changes in energy demands and undergoing fission and fusion, and mitophagy [107]. Intracellular localization of mitochondria corresponds to sites of ATP consumption. Mitochondria are located in close proximity to myofibrils in skeletal muscle and cardiac myocytes. In neurons, mitochondria are trafficked along microtubules in axons to synapses where ATP is necessary for neuronal transmission. Fusion and fission
SS-31 repairs cellular structure and restore function during aging
A hallmark of cellular aging is the breakdown of the cytoskeleton and loss of cell structure. The assembly of actin filaments is ATP-dependent and decline in cellular ATP leads to rearrangement of the actin cytoskeleton, loss of cell polarity and cell-cell contact, and internalization of cell surface proteins [112,113]. During acute renal ischemia, there is loss of cell-cell contact and detachment of proximal tubular cells due to redistribution of E-cadherin and β1-integrin from the lateral and
SS-31 prevents cell death and inflammation
Oxidized cardiolipin can translocate from the IMM to the OMM. Its appearance on the OMM serves as a danger signal to trigger a number of cellular stress responses [116]. Oxidized cardiolipin on the OMM triggers mitochondrial fission and the recruitment of endoplasmic reticulum membranes to initiate mitophagy to eliminate damaged mitochondria [117,118]. Oxidized cardiolipin on the OMM can cause the release of cytochrome c from mitochondria to initiate apoptosis [119,120]. Finally, oxidized
SS-31 boosts the body's natural ability to heal itself
Survival of any living organism critically depends on its ability to repair and regenerate damaged tissues and/or organs during its lifetime following injury, disease, or aging. Post-injury regeneration of skeletal muscle occurs through activation, proliferation, and differentiation of resident myogenic stem cells. Skeletal muscle is highly vascularized, and muscle regeneration requires interaction between endothelial precursor cells and myogenic precursor cells [139]. Quiescent stem cells have
Summary
Serendipity played a pivotal role in the discovery of these cardiolipin-targeted peptides. These peptides would never have been discovered by rational drug design because mitochondrial production of ATP requires the concerted effort of so many proteins in the ETC. Rather than target a single protein, these aromatic-cationic peptides target cardiolipin to induce structural change in the membrane and modify the spatial organization of all ETC proteins to optimize electron transfer. Such a
Conflicts of interest
The author (HHS) is the inventor of the cardiolipin-targeted peptides described in this article, and the Founder of Stealth Biotherapeutics, a clinical stage biopharmaceutical company that licensed this peptide technology from the Cornell Research Foundation for research and development.
Acknowledgements
We thank Peter Rabinovitch, David Marcinek and Kevin Conley for their collaboration on aging studies over the years. Their insight has contributed immensely to our understanding of the action of these cardiolipin-targeted compounds. Our work has been supported by the National Institute on Aging (PO1 AG001751) and the Social Profit Network.