Hyperactive mTORC1/4EBP1 Signaling Dysregulates Proteostasis and Accelerates Cardiac Aging.

The mechanistic target of rapamycin complex 1 (mTORC1) has a major impact on aging by regulation of proteostasis. It is well established that mTORC1 signaling is hyperactivated with aging and age-related diseases. Previous studies have shown that partial inhibition of mTOR signaling by rapamycin reverses the age-related decline in cardiac function and structure in old mice. However, the downstream signaling pathways involved in this protection against cardiac aging have not been established. TORC1 phosphorylates 4E-binding protein 1 (4EBP1) to promote the initiation of cap-dependent translation. The aim of this project is to examine the role of the mTORC1/4EBP1 axis in age-related cardiac dysfunction. We utilized a whole-body 4EBP1 KO mouse model, which mimics a hyperactive 4EBP1/eIF4E axis, to investigate the effects of hyperactive mTORC1/4EBP1 axis in cardiac aging. Echocardiographic measurements revealed that young 4EBP1 KO mice have no difference in cardiac function at baseline compared to WT mice. Interestingly, middle-aged (14-15-month-old) 4EBP1 KO mice show impaired diastolic function and myocardial performance compared to age-matched WT mice and their diastolic function and myocardial performance are at similar levels as 24-month-old WT mice, suggesting that 4EBP1 KO mice experience accelerated cardiac aging. Old 4EBP1 KO mice show further declines in systolic and diastolic function compared to middle-aged 4EBP1 KO mice and have worse systolic and diastolic function than age-matched old WT mice. Gene expression levels of heart failure markers are not different between 4EBP1 KO and WT mice at these advanced ages. However, ribosomal biogenesis and overall protein ubiquitination are significantly increased in 4EBP1 KO mice when compared to WT, which suggests dysregulated proteostasis. Together, these results show that a hyperactive 4EBP1/eIF4E axis accelerates cardiac aging, potentially by dysregulating proteostasis.

Late-life Rapamycin Treatment Enhances Cardiomyocyte Relaxation Kinetics and Reduces Myocardial Stiffness.

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.

An In Vivo Stable Isotope Labeling Method to Investigate Individual Matrix Protein Synthesis, Ribosomal Biogenesis, and Cellular Proliferation in Murine Articular Cartilage.

Targeting chondrocyte dynamics is a strategy for slowing osteoarthritis progression during aging. We describe a stable-isotope method using in vivo deuterium oxide labeling and mass spectrometry to measure protein concentration, protein half-life, cell proliferation, and ribosomal biogenesis in a single sample of murine articular cartilage. We hypothesized that a 60-d labeling period would capture age-related declines in cartilage matrix protein content, protein synthesis rates, and cellular proliferation. Knee cartilage was harvested to the subchondral bone from 25- to 90-wk-old female C57BL/6J mice treated with deuterium oxide for 15, 30, 45, and 60 d. We measured protein concentration and half-lives using targeted high resolution accurate mass spectrometry and d2ome data processing software. Deuterium enrichment was quantified in isolated DNA and RNA to measure cell proliferation and ribosomal biogenesis, respectively. Most collagen isoforms were less abundant in aged animals, with negligible collagen synthesis at either age. In contrast, age altered the concentration and half-lives of many proteoglycans and other matrix proteins, including several with greater concentration and half-lives in older mice such as proteoglycan 4, clusterin, and fibronectin-1. Cellular proteins were less abundant in older animals, consistent with reduced cellularity. Nevertheless, deuterium was maximally incorporated into 60% of DNA and RNA by 15 d of labeling in both age groups, suggesting the presence of two large pools of either rapidly (<15 d) or slowly (>60 d) proliferating cells. Our findings indicate that age-associated changes in cartilage matrix protein content and synthesis occur without detectable changes in the relative number of proliferating cells.

Determining the contributions of protein synthesis and breakdown to muscle atrophy requires non-steady-state equations.

BACKGROUND: Ageing and cachexia cause a loss of muscle mass over time, indicating that protein breakdown exceeds protein synthesis. Deuterium oxide (D2 O) is used for studies of protein turnover because of the advantages of long-term labelling, but these methods introduce considerations that have been largely overlooked when studying conditions of protein gain or loss. The purpose of this study was to demonstrate the importance of accounting for a change in protein mass, a non-steady state, during D2 O labelling studies while also exploring the contribution of protein synthesis and breakdown to denervation-induced muscle atrophy. METHODS: Adult (6 months) male C57BL/6 mice (n = 14) were labelled with D2 O for a total of 7 days following unilateral sciatic nerve transection to induce denervation of hindlimb muscles. The contralateral sham limb and nonsurgical mice (n = 5) were used as two different controls to account for potential crossover effects of denervation. We calculated gastrocnemius myofibrillar and collagen protein synthesis and breakdown assuming steady-state or using non-steady-state modelling. We measured RNA synthesis rates to further understand ribosomal turnover during atrophy. RESULTS: Gastrocnemius mass was less in denervated muscle (137 ± 9 mg) compared with sham (174 ± 15 mg; P < 0.0001) or nonsurgical control (162 ± 5 mg; P < 0.0001). With steady-state calculations, fractional synthesis and breakdown rates (FSR and FBR) were lower in the denervated muscle (1.49 ± 0.06%/day) compared with sham (1.81 ± 0.09%/day; P < 0.0001) or nonsurgical control (2.27 ± 0.04%/day; P < 0.0001). When adjusting for change in protein mass, FSR was 4.21 ± 0.19%/day in denervated limb, whereas FBR was 4.09 ± 0.22%/day. When considering change in protein mass (ksyn ), myofibrillar synthesis was lower in denervated limb (2.44 ± 0.14 mg/day) compared with sham (3.43 ± 0.22 mg/day; P < 0.0001) and non-surgical control (3.74 ± 0.12 mg/day; P < 0.0001), whereas rate of protein breakdown (kdeg, 1/t) was greater in denervated limb (0.050 ± 0.003) compared with sham (0.019 ± 0.001; P < 0.0001) and nonsurgical control (0.023 ± 0.000; P < 0.0001). Muscle collagen breakdown was completely inhibited during denervation. There was a strong correlation (r = 0.83, P < 0.001) between RNA and myofibrillar protein synthesis in sham but not denervated muscle. CONCLUSIONS: We show conflicting results between steady- and non-steady-state calculations on myofibrillar protein synthesis and breakdown during periods of muscle loss. We also found that collagen accumulation was largely from a decrease in collagen breakdown. Comparison between sham and non-surgical control demonstrated a crossover effect of denervation on myofibrillar protein synthesis and ribosomal biogenesis, which impacts study design for unilateral atrophy studies. These considerations are important because not accounting for them can mislead therapeutic attempts to maintain muscle mass.

A Novel Stable Isotope Approach Demonstrates Surprising Degree of Age-Related Decline in Skeletal Muscle Collagen Proteostasis.

Age-related deterioration in turnover of collagen proteins accelerates extracellular matrix fibrosis and hinders adaptation to external stimuli. This project sought to understand factors that increase skeletal muscle fibrosis with age by studying what we term the dynamic protein pool. We hypothesized that the dynamic protein pool size of muscle collagen decreases with age, thus indicating a decrease in proteostatic maintenance (ie, ability to maintain proteostasis), and that failure to account for these changes impacts the interpretation of tracer-measured synthesis rates. We used deuterium oxide (D2O) labeling for up to 60 days in adult (6 months) and old (23 months) mice. The dynamic protein pool in adult skeletal muscle was 65% in tibialis anterior (TA), but only 28% in gastrocnemius (Gastroc). In aged muscle, the dynamic protein pool was further decreased to only 35% and 14% for TA and Gastroc, respectively. We showed that this loss in dynamic pool size was associated with increases in markers of fibrosis and decreased proteostatic maintenance. We demonstrate that aged muscle has higher rates of collagen protein synthesis and lower rates of collagen protein breakdown, which causes collagen accumulation. We further demonstrated that the normal assumption of complete protein renewal and the standard practice of taking a single sample with isotope labeling have profound impacts on interpretation of the genesis of fibrosis. Strategies to maintain muscle function with aging should focus on the dynamic protein pool with attention to methodological strategies to assess those changes.

Primary Human Cardiomyocytes and Cardiofibroblasts Treated with Sera from Myocarditis Patients Exhibit an Increased Iron Demand and Complex Changes in the Gene Expression.

Cardiac fibroblasts and cardiomyocytes are the main cells involved in the pathophysiology of myocarditis (MCD). These cells are especially sensitive to changes in iron homeostasis, which is extremely important for the optimal maintenance of crucial cellular processes. However, the exact role of iron status in the pathophysiology of MCD remains unknown. We cultured primary human cardiomyocytes (hCM) and cardiofibroblasts (hCF) with sera from acute MCD patients and healthy controls to mimic the effects of systemic inflammation on these cells. Next, we performed an initial small-scale (n = 3 per group) RNA sequencing experiment to investigate the global cellular response to the exposure on sera. In both cell lines, transcriptomic data analysis revealed many alterations in gene expression, which are related to disturbed canonical pathways and the progression of cardiac diseases. Moreover, hCM exhibited changes in the iron homeostasis pathway. To further investigate these alterations in sera-treated cells, we performed a larger-scale (n = 10 for controls, n = 18 for MCD) follow-up study and evaluated the expression of genes involved in iron metabolism. In both cell lines, we demonstrated an increased expression of transferrin receptor 1 (TFR1) and ferritin in MCD serum-treated cells as compared to controls, suggesting increased iron demand. Furthermore, we related TFR1 expression with the clinical profile of patients and showed that greater iron demand in sera-treated cells was associated with higher inflammation score (interleukin 6 (IL-6), C-reactive protein (CRP)) and advanced neurohormonal activation (NT-proBNP) in patients. Collectively, our data suggest that the malfunctioning of cardiomyocytes and cardiofibroblasts in the course of MCD might be related to alterations in the iron homeostasis.

Structural and functional abnormalities in iron-depleted heart.

Iron deficiency (ID) is a common and ominous comorbidity in heart failure (HF) and predicts worse outcomes, independently of the presence of anaemia. Accumulated data from animal models of systemic ID suggest that ID is associated with several functional and structural abnormalities of the heart. However, the exact role of myocardial iron deficiency irrespective of systemic ID and/or anaemia has been elusive. Recently, several transgenic models of cardiac-specific ID have been developed to investigate the influence of ID on cardiac tissue. In this review, we discuss structural and functional cardiac consequences of ID in these models and summarize data from clinical studies. Moreover, the beneficial effects of intravenous iron supplementation are specified.

Iron Depletion Affects Genes Encoding Mitochondrial Electron Transport Chain and Genes of Non-Oxidative Metabolism, Pyruvate Kinase and Lactate Dehydrogenase, in Primary Human Cardiac Myocytes Cultured upon Mechanical Stretch.

(1) Background: Oxidative energy metabolism is presumed to rely on the optimal iron supply. Primary human cardiac myocytes (HCM) exposed to different iron availability conditions during mechanical stretch are anticipated to demonstrate expression changes of genes involved in aerobic and anaerobic metabolic pathways. (2) Methods: HCM were cultured for 48 h either in static conditions and upon mechanical stretch at the optimal versus reduced versus increased iron concentrations. We analyzed the expression of pyruvate kinase (PKM2), lactate dehydrogenase A (LDHA), and mitochondrial complexes I⁻V at the mRNA and protein levels. The concentration of l-lactate was assessed by means of lactate oxidase method-based kit. (3) Results: Reduced iron concentrations during mechanical work caused a decreased expression of complexes I⁻V (all p < 0.05). The expression of PKM2 and LDHA, as well as the medium concentration of l-lactate, was increased in these conditions (both p < 0.05). HCM exposed to the increased iron concentration during mechanical effort demonstrated a decreased expression of mitochondrial complexes (all p < 0.01); however, a decrement was smaller than in case of iron chelation (p < 0.05). The iron-enriched medium caused a decrease in expression of LDHA and did not influence the concentration of l-lactate. (4) Conclusions: During mechanical effort, the reduced iron availability enhances anaerobic glycolysis and extracellular lactate production, whilst decreasing mitochondrial aerobic pathway in HCM. Iron enrichment during mechanical effort may be protective in the context of intracellular protein machinery of non-oxidative metabolism with no effect on the extracellular lactate concentration.