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Diastolic dysfunction is a key feature of the aging heart. We have shown that late-life treatment with mTOR inhibitor, rapamycin, reverses age-related diastolic dysfunction in mice but the molecular mechanisms of the reversal remain unclear. To dissect the mechanisms by which rapamycin improves diastolic function in old mice, we examined the effects of rapamycin treatment at the levels of single cardiomyocyte, myofibril and multicellular cardiac muscle. Compared to young cardiomyocytes, isolated cardiomyocytes from old control mice exhibited prolonged time to 90% relaxation (RT (90) ) and time to 90% Ca (2+) transient decay (DT (90) ), indicating slower relaxation kinetics and calcium reuptake with age. Late-life rapamycin treatment for 10 weeks completely normalized RT (90) and partially normalized DT (90) , suggesting improved Ca (2+) handling contributes partially to the rapamycin-induced improved cardiomyocyte relaxation. In addition, rapamycin treatment in old mice enhanced the kinetics of sarcomere shortening and Ca (2+) transient increase in old control cardiomyocytes. Myofibrils from old rapamycin-treated mice displayed increased rate of the fast, exponential decay phase of relaxation compared to old controls. The improved myofibrillar kinetics were accompanied by an increase in MyBP-C phosphorylation at S282 following rapamycin treatment. We also showed that late-life rapamycin treatment normalized the age-related increase in passive stiffness of demembranated cardiac trabeculae through a mechanism independent of titin isoform shift. In summary, our results showed that rapamycin treatment normalizes the age-related impairments in cardiomyocyte relaxation, which works conjointly with reduced myocardial stiffness to reverse age-related diastolic dysfunction.

Background: Disruption of metabolic and bioenergetic homeostasis related to mitochondrial dysfunction is a key driver of aging biology. Therefore, targeting mitochondrial function would be a rational approach to slowing aging. Elamipretide (Elam, a.k.a. SS-31) is a peptide known to target mitochondria and suppress mammalian signs of aging. The present study was designed to examine the phenotypic effects of long-term Elam treatment on aging in C57BL/6 mice starting at 18 months of age. Methods: Mice were fed regular chow (RC diet) or a diet high in fat and sugar (HF diet) and treated with 3 mg/ kg of Elam or saline subcutaneously 5 days per week for 10 months. Physiological performance assessments were conducted at 28 months of age. Results: Elam improved the physical performance of males but not females, while in females Elam improved cognitive performance and enhanced the maintenance of body weight and fat mass. It also improved diastolic function in both males and females, but to a greater extent in males. The HF diet over 10 months had a negative effect on health span, as it increased body fat and decreased muscle strength and heart function, especially in females. Conclusions: Elam enhanced healthy aging and cardiac function in both male and female mice, although the specific effects on function differed between sexes. In females, the treatment led to better cognitive performance and maintenance of body composition, while in males, performance on a rotating rod was preserved. These overall observations have translational implications for considering additional studies using Elam in therapeutic or preventive approaches for aging and age-related diseases. Keywords: Aging, mitochondria, elamipretide, C57BL/6 mice, high fat and sugar diet

The mitochondrial-rich renal tubule cells are key regulators of blood homeostasis via excretion and reabsorption of metabolic waste. With age, tubules are subject to increasing mitochondrial dysfunction and declining nicotinamide adenine dinucleotide (NAD+) levels, both hampering ATP production efficiency. We tested two mitochondrial interventions in young (6-mo) and aged (26-mo) adult male mice: elamipretide (ELAM), a tetrapeptide in clinical trials that improves mitochondrial structure and function, and nicotinamide mononucleotide (NMN), an NAD+ intermediate and commercially available oral supplement. Kidneys were analyzed from young and aged mice after eight weeks of treatment with ELAM (3 mg/kg/day), NMN (300 mg/kg/day), or from aged mice treated with the two interventions combined (ELAM+NMN). We hypothesized that combining pharmacologic treatments to ameliorate mitochondrial dysfunction and boost NAD+ levels, would more effectively reduce kidney aging than either intervention alone. Unexpectedly, in aged kidneys, NMN increased expression of genetic markers of inflammation (IL-1-beta; and Ccl2) and tubule injury (Kim-1). Metabolomics of endpoint sera showed that NMN-treated aged mice had higher circulating levels of uremic toxins than either aged controls or young NMN-treated mice. ELAM+NMN-treated aged mice accumulated uremic toxins like NMN-only aged mice, but reduced IL-1-beta; and Ccl2 kidney mRNA. This suggests that pre-existing mitochondrial dysfunction in aged kidney underlies susceptibility to inflammatory signaling with NMN supplementation in aged, but not young, mice. These findings demonstrate age and tissue dependent effects on downstream metabolic accumulation from NMN and highlight the need for targeted analysis of aged kidneys to assess the safety of anti-aging supplements in older populations.

Diastolic dysfunction is a key feature of the aging heart. We have shown that late-life treatment with mTOR inhibitor, rapamycin, reverses age-related diastolic dysfunction in mice but the molecular mechanisms of the reversal remain unclear. To dissect the mechanisms by which rapamycin improves diastolic function in old mice, we examined the effects of rapamycin treatment at the levels of single cardiomyocyte, myofibril and multicellular cardiac muscle. Compared to young cardiomyocytes, isolated cardiomyocytes from old control mice exhibited prolonged time to 90% relaxation (RT 90 ) and time to 90% Ca 2+ transient decay (DT 90 ), indicating slower relaxation kinetics and calcium reuptake with age. Late-life rapamycin treatment for 10 weeks completely normalized RT 90 and partially normalized DT 90 , suggesting improved Ca 2+ handling contributes partially to the rapamycin-induced improved cardiomyocyte relaxation. In addition, rapamycin treatment in old mice enhanced the kinetics of sarcomere shortening and Ca 2+ transient increase in old control cardiomyocytes. Myofibrils from old rapamycin-treated mice displayed increased rate of the fast, exponential decay phase of relaxation compared to old controls. The improved myofibrillar kinetics were accompanied by an increase in MyBP-C phosphorylation at S282 following rapamycin treatment. We also showed that late-life rapamycin treatment normalized the age-related increase in passive stiffness of demembranated cardiac trabeculae through a mechanism independent of titin isoform shift. In summary, our results showed that rapamycin treatment normalizes the age-related impairments in cardiomyocyte relaxation, which works conjointly with reduced myocardial stiffness to reverse age-related diastolic dysfunction.

Aging muscle experiences functional decline in part mediated by impaired mitochondrial ADP sensitivity. Elamipretide (ELAM) rapidly improves physiological and mitochondrial function in aging and binds directly to the mitochondrial ADP transporter ANT. We hypothesized that ELAM improves ADP sensitivity in aging leading to rescued physiological function. We measured the response to ADP stimulation in young and old muscle mitochondria with ELAM treatment, in vivo heart and muscle function, and compared protein abundance, phosphorylation, and S-glutathionylation of ADP/ATP pathway proteins. ELAM treatment increased ADP sensitivity in old muscle mitochondria by increasing uptake of ADP through the ANT and rescued muscle force and heart systolic function. Protein abundance in the ADP/ATP transport and synthesis pathway was unchanged, but ELAM treatment decreased protein s-glutathionylation incuding of ANT. Mitochondrial ADP sensitivity is rapidly modifiable. This research supports the hypothesis that ELAM improves ANT function in aging and links mitochondrial ADP sensitivity to physiological function.