Stem cell secretome treatment improves whole-body metabolism, reduces adiposity, and promotes skeletal muscle function in aged mice.

Aging coincides with the progressive loss of muscle mass and strength, increased adiposity, and diminished physical function. Accordingly, interventions aimed at improving muscle, metabolic, and/or physical health are of interest to mitigate the adverse effects of aging. In this study, we tested a stem cell secretome product, which contains extracellular vesicles and growth, cytoskeletal remodeling, and immunomodulatory factors. We examined the effects of 4 weeks of 2×/week unilateral intramuscular secretome injections (quadriceps) in ambulatory aged male C57BL/6 mice (22-24 months) compared to saline-injected aged-matched controls. Secretome delivery substantially increased whole-body lean mass and decreased fat mass, corresponding to higher myofiber cross-sectional area and smaller adipocyte size, respectively. Secretome-treated mice also had greater whole-body physical function (grip strength and rotarod performance) and had higher energy expenditure and physical activity levels compared to control mice. Furthermore, secretome-treated mice had greater skeletal muscle Pax7+ cell abundance, capillary density, collagen IV turnover, reduced intramuscular lipids, and greater Akt and hormone sensitive lipase phosphorylation in adipose tissue. Finally, secretome treatment in vitro directly enhanced muscle cell growth and IL-6 production, and in adipocytes, it reduced lipid content and improved insulin sensitivity. Moreover, indirect treatment with secretome-treated myotube culture media also enhanced muscle cell growth and adipocyte size reduction. Together, these data suggest that intramuscular treatment with a stem cell secretome improves whole-body metabolism, physical function, and remodels skeletal muscle and adipose tissue in aged mice.

Sequestosome 1 (p62) mitigates hypoxia-induced cardiac dysfunction by stabilizing hypoxia-inducible factor 1α and nuclear factor erythroid 2-related factor 2.

AIMS: Heart failure due to ischaemic heart disease (IHD) is a leading cause of mortality worldwide. A major contributing factor to IHD-induced cardiac damage is hypoxia. Sequestosome 1 (p62) is a multi-functional adaptor protein with pleiotropic roles in autophagy, proteostasis, inflammation, and cancer. Despite abundant expression in cardiomyocytes, the role of p62 in cardiac physiology is not well understood. We hypothesized that cardiomyocyte-specific p62 deletion evokes hypoxia-induced cardiac pathology by impairing hypoxia-inducible factor 1α (Hif-1α) and nuclear factor erythroid 2-related factor 2 (Nrf2) signalling. METHODS AND RESULTS: Adult mice with germline deletion of cardiomyocyte p62 exhibited mild cardiac dysfunction under normoxic conditions. Transcriptomic analyses revealed a selective impairment in Nrf2 target genes in the hearts from these mice. Demonstrating the functional importance of this adaptor protein, adult mice with inducible depletion of cardiomyocyte p62 displayed hypoxia-induced contractile dysfunction, oxidative stress, and cell death. Mechanistically, p62-depleted hearts exhibit impaired Hif-1α and Nrf2 transcriptional activity. Because findings from these two murine models suggested a cardioprotective role for p62, mechanisms were evaluated using H9c2 cardiomyoblasts. Loss of p62 in H9c2 cells exposed to hypoxia reduced Hif-1α and Nrf2 protein levels. Further, the lack of p62 decreased Nrf2 protein expression, nuclear translocation, and transcriptional activity. Repressed Nrf2 activity associated with heightened Nrf2-Keap1 co-localization in p62-deficient cells, which was concurrent with increased Nrf2 ubiquitination facilitated by the E3 ligase Cullin 3, followed by proteasomal-mediated degradation. Substantiating our results, a gain of p62 in H9c2 cells stabilized Nrf2 and increased the transcriptional activity of Nrf2 downstream targets. CONCLUSION: Cardiac p62 mitigates hypoxia-induced cardiac dysfunction by stabilizing Hif-1α and Nrf2.

Control of NAD+ homeostasis by autophagic flux modulates mitochondrial and cardiac function.

Impaired autophagy is known to cause mitochondrial dysfunction and heart failure, in part due to altered mitophagy and protein quality control. However, whether additional mechanisms are involved in the development of mitochondrial dysfunction and heart failure in the setting of deficient autophagic flux remains poorly explored. Here, we show that impaired autophagic flux reduces nicotinamide adenine dinucleotide (NAD+) availability in cardiomyocytes. NAD+ deficiency upon autophagic impairment is attributable to the induction of nicotinamide N-methyltransferase (NNMT), which methylates the NAD+ precursor nicotinamide (NAM) to generate N-methyl-nicotinamide (MeNAM). The administration of nicotinamide mononucleotide (NMN) or inhibition of NNMT activity in autophagy-deficient hearts and cardiomyocytes restores NAD+ levels and ameliorates cardiac and mitochondrial dysfunction. Mechanistically, autophagic inhibition causes the accumulation of SQSTM1, which activates NF-κB signaling and promotes NNMT transcription. In summary, we describe a novel mechanism illustrating how autophagic flux maintains mitochondrial and cardiac function by mediating SQSTM1-NF-κB-NNMT signaling and controlling the cellular levels of NAD+.

Mitofusin-2 Regulates Platelet Mitochondria and Function.

BACKGROUND: Single-nucleotide polymorphisms linked with the rs1474868 T allele (MFN2 [mitofusin-2] T/T) in the human mitochondrial fusion protein MFN2 gene are associated with reduced platelet MFN2 RNA expression and platelet counts. This study investigates the impact of MFN2 on megakaryocyte and platelet biology. METHODS: Mice with megakaryocyte/platelet deletion of Mfn2 (Mfn2-/- [Mfn2 conditional knockout]) were generated using Pf4-Cre crossed with floxed Mfn2 mice. Human megakaryocytes were generated from cord blood and platelets isolated from healthy subjects genotyped for rs1474868. Ex vivo approaches assessed mitochondrial morphology, function, and platelet activation responses. In vivo measurements included endogenous/transfused platelet life span, tail bleed time, transient middle cerebral artery occlusion, and pulmonary vascular permeability/hemorrhage following lipopolysaccharide-induced acute lung injury. RESULTS: Mitochondria was more fragmented in megakaryocytes derived from Mfn2-/- mice and from human cord blood with MFN2 T/T genotype compared with control megakaryocytes. Human resting platelets of MFN2 T/T genotype had reduced MFN2 protein, diminished mitochondrial membrane potential, and an increased rate of phosphatidylserine exposure during ex vivo culture. Platelet counts and platelet life span were reduced in Mfn2-/- mice accompanied by an increased rate of phosphatidylserine exposure in resting platelets, especially aged platelets, during ex vivo culture. Mfn2-/- also decreased platelet mitochondrial membrane potential (basal) and activated mitochondrial oxygen consumption rate, reactive oxygen species generation, calcium flux, platelet-neutrophil aggregate formation, and phosphatidylserine exposure following dual agonist activation. Ultimately, Mfn2-/- mice showed prolonged tail bleed times, decreased ischemic stroke infarct size after cerebral ischemia-reperfusion, and exacerbated pulmonary inflammatory hemorrhage following lipopolysaccharide-induced acute lung injury. Analysis of MFN2 SNPs in the iSPAAR study (Identification of SNPs Predisposing to Altered ALI Risk) identified a significant association between MFN2 and 28-day mortality in patients with acute respiratory distress syndrome. CONCLUSIONS: Mfn2 preserves mitochondrial phenotypes in megakaryocytes and platelets and influences platelet life span, function, and outcomes of stroke and lung injury.

Nonsense Variant PRDM16-Q187X Causes Impaired Myocardial Development and TGF-ß Signaling Resulting in Noncompaction Cardiomyopathy in Humans and Mice.

BACKGROUND: PRDM16 plays a role in myocardial development through TGF-ß (transforming growth factor-beta) signaling. Recent evidence suggests that loss of PRDM16 expression is associated with cardiomyopathy development in mice, although its role in human cardiomyopathy development is unclear. This study aims to determine the impact of PRDM16 loss-of-function variants on cardiomyopathy in humans. METHODS: Individuals with PRDM16 variants were identified and consented. Induced pluripotent stem cell-derived cardiomyocytes were generated from a proband hosting a Q187X nonsense variant as an in vitro model and underwent proliferative and transcriptional analyses. CRISPR (clustered regularly interspaced short palindromic repeats)-mediated knock-in mouse model hosting the Prdm16Q187X allele was generated and subjected to ECG, histological, and transcriptional analysis. RESULTS: We report 2 probands with loss-of-function PRDM16 variants and pediatric left ventricular noncompaction cardiomyopathy. One proband hosts a PRDM16-Q187X variant with left ventricular noncompaction cardiomyopathy and demonstrated infant-onset heart failure, which was selected for further study. Induced pluripotent stem cell-derived cardiomyocytes prepared from the PRDM16-Q187X proband demonstrated a statistically significant impairment in myocyte proliferation and increased apoptosis associated with transcriptional dysregulation of genes implicated in cardiac maturation, including TGF-ß-associated transcripts. Homozygous Prdm16Q187X/Q187X mice demonstrated an underdeveloped compact myocardium and were embryonically lethal. Heterozygous Prdm16Q187X/WT mice demonstrated significantly smaller ventricular dimensions, heightened fibrosis, and age-dependent loss of TGF-ß expression. Mechanistic studies were undertaken in H9c2 cardiomyoblasts to show that PRDM16 binds TGFB3 promoter and represses its transcription. CONCLUSIONS: Novel loss-of-function PRDM16 variant impairs myocardial development resulting in noncompaction cardiomyopathy in humans and mice associated with altered TGF-ß signaling.

PRDM16 Deletion Is Associated With Sex-dependent Cardiomyopathy and Cardiac Mortality: A Translational, Multi-Institutional Cohort Study.

BACKGROUND: 1p36 deletion syndrome can predispose to pediatric-onset cardiomyopathy. Deletion breakpoints are variable and may delete the transcription factor PRDM16. Early studies suggest that deletion of PRDM16 may underlie cardiomyopathy in patients with 1p36 deletion; however, the prognostic impact of PRDM16 loss is unknown. METHODS: This retrospective cohort included subjects with 1p36 deletion syndrome from 4 hospitals. Prevalence of cardiomyopathy and freedom from death, cardiac transplantation, or ventricular assist device were analyzed. A systematic review cohort was derived for further analysis. A cardiac-specific Prdm16 knockout mouse (Prdm16 conditional knockout) was generated. Echocardiography was performed at 4 and 6 to 7 months. Histology staining and qPCR were performed at 7 months to assess fibrosis. RESULTS: The retrospective cohort included 71 patients. Among individuals with PRDM16 deleted, 34.5% developed cardiomyopathy versus 7.7% of individuals with PRDM16 not deleted (P=0.1). In the combined retrospective and systematic review cohort (n=134), PRDM16 deletion-associated cardiomyopathy risk was recapitulated and significant (29.1% versus 10.8%, P=0.03). PRDM16 deletion was associated with increased risk of death, cardiac transplant, or ventricular assist device (P=0.04). Among those PRDM16 deleted, 34.5% of females developed cardiomyopathy versus 16.7% of their male counterparts (P=0.2). We find sex-specific differences in the incidence and the severity of contractile dysfunction and fibrosis in female Prdm16 conditional knockout mice. Further, female Prdm16 conditional knockout mice demonstrate significantly elevated risk of mortality (P=0.0003). CONCLUSIONS: PRDM16 deletion is associated with a significantly increased risk of cardiomyopathy and cardiac mortality. Prdm16 conditional knockout mice develop cardiomyopathy in a sex-biased way. Patients with PRDM16 deletion should be assessed for cardiac disease.

BMPER is a marker of adipose progenitors and adipocytes and a positive modulator of adipogenesis.

Autocrine and paracrine signaling regulating adipogenesis in white adipose tissue remains largely unclear. Here we used single-cell RNA-sequencing (RNA-seq) and single nuclei RNA-sequencing (snRNA-seq) to identify markers of adipose progenitor cells (APCs) and adipogenic modulators in visceral adipose tissue (VAT) of humans and mice. Our study confirmed the presence of major cellular clusters in humans and mice and established important sex and diet-specific dissimilarities in cell proportions. Here we show that bone morphogenetic protein (BMP)-binding endothelial regulator (BMPER) is a conserved marker for APCs and adipocytes in VAT in humans and mice. Further, BMPER is highly enriched in lineage negative stromal vascular cells and its expression is significantly higher in visceral compared to subcutaneous APCs in mice. BMPER expression and release peaked by day four post-differentiation in 3T3-L1 preadipocytes. We reveal that BMPER is required for adipogenesis both in 3T3-L1 preadipocytes and in mouse APCs. Together, this study identified BMPER as a positive modulator of adipogenesis.

SMYD1a protects the heart from ischemic injury by regulating OPA1-mediated cristae remodeling and supercomplex formation.

SMYD1, a striated muscle-specific lysine methyltransferase, was originally shown to play a key role in embryonic cardiac development but more recently we demonstrated that loss of Smyd1 in the murine adult heart leads to cardiac hypertrophy and failure. However, the effects of SMYD1 overexpression in the heart and its molecular function in the cardiomyocyte in response to ischemic stress are unknown. In this study, we show that inducible, cardiomyocyte-specific overexpression of SMYD1a in mice protects the heart from ischemic injury as seen by a > 50% reduction in infarct size and decreased myocyte cell death. We also demonstrate that attenuated pathological remodeling is a result of enhanced mitochondrial respiration efficiency, which is driven by increased mitochondrial cristae formation and stabilization of respiratory chain supercomplexes within the cristae. These morphological changes occur concomitant with increased OPA1 expression, a known driver of cristae morphology and supercomplex formation. Together, these analyses identify OPA1 as a novel downstream target of SMYD1a whereby cardiomyocytes upregulate energy efficiency to dynamically adapt to the energy demands of the cell. In addition, these findings highlight a new epigenetic mechanism by which SMYD1a regulates mitochondrial energetics and functions to protect the heart from ischemic injury.

Lipid hydroperoxides promote sarcopenia through carbonyl stress.

Reactive oxygen species (ROS) accumulation is a cardinal feature of skeletal muscle atrophy. ROS refers to a collection of radical molecules whose cellular signals are vast, and it is unclear which downstream consequences of ROS are responsible for the loss of muscle mass and strength. Here, we show that lipid hydroperoxides (LOOH) are increased with age and disuse, and the accumulation of LOOH by deletion of glutathione peroxidase 4 (GPx4) is sufficient to augment muscle atrophy. LOOH promoted atrophy in a lysosomal-dependent, proteasomal-independent manner. In young and old mice, genetic and pharmacological neutralization of LOOH or their secondary reactive lipid aldehydes robustly prevented muscle atrophy and weakness, indicating that LOOH-derived carbonyl stress mediates age- and disuse-induced muscle dysfunction. Our findings provide novel insights for the role of LOOH in sarcopenia including a therapeutic implication by pharmacological suppression.

The dynamic interplay between cardiac mitochondrial health and myocardial structural remodeling in metabolic heart disease, aging, and heart failure.

This review provides a holistic perspective on the bi-directional relationship between cardiac mitochondrial dysfunction and myocardial structural remodeling in the context of metabolic heart disease, natural cardiac aging, and heart failure. First, a review of the physiologic and molecular drivers of cardiac mitochondrial dysfunction across a range of increasingly prevalent conditions such as metabolic syndrome and cardiac aging is presented, followed by a general review of the mechanisms of mitochondrial quality control (QC) in the heart. Several important mechanisms by which cardiac mitochondrial dysfunction triggers or contributes to structural remodeling of the heart are discussed: accumulated metabolic byproducts, oxidative damage, impaired mitochondrial QC, and mitochondrial-mediated cell death identified as substantial mechanistic contributors to cardiac structural remodeling such as hypertrophy and myocardial fibrosis. Subsequently, the less studied but nevertheless important reverse relationship is explored: the mechanisms by which cardiac structural remodeling feeds back to further alter mitochondrial bioenergetic function. We then provide a condensed pathogenesis of several increasingly important clinical conditions in which these relationships are central: diabetic cardiomyopathy, age-associated declines in cardiac function, and the progression to heart failure, with or without preserved ejection fraction. Finally, we identify promising therapeutic opportunities targeting mitochondrial function in these conditions.

AAV-based gene therapy prevents and halts the progression of dilated cardiomyopathy in a mouse model of phosphoglucomutase 1 deficiency (PGM1-CDG).

Phosphoglucomutase 1 (PGM1) deficiency is recognized as the third most common N-linked congenital disorders of glycosylation (CDG) in humans. Affected individuals present with liver, musculoskeletal, endocrine, and coagulation symptoms; however, the most life-threatening complication is the early onset of dilated cardiomyopathy (DCM). Recently, we discovered that oral D-galactose supplementation improved liver disease, endocrine, and coagulation abnormalities, but does not alleviate the fatal cardiomyopathy and the associated myopathy. Here we report on left ventricular ejection fraction (LVEF) in 6 individuals with PGM1-CDG. LVEF was pathologically low in most of these individuals and varied between 10% and 65%. To study the pathobiology of the cardiac disease observed in PGM1-CDG, we constructed a novel cardiomyocyte-specific conditional Pgm2 gene (mouse ortholog of human PGM1) knockout (Pgm2 cKO) mouse model. Echocardiography studies corroborated a DCM phenotype with significantly reduced ejection fraction and left ventricular dilation similar to those seen in individuals with PGM1-CDG. Histological studies demonstrated excess glycogen accumulation and fibrosis, while ultrastructural analysis revealed Z-disk disarray and swollen/fragmented mitochondria, which was similar to the ultrastructural pathology in the cardiac explant of an individual with PGM1-CDG. In addition, we found decreased mitochondrial function in the heart of KO mice. Transcriptomic analysis of hearts from mutant mice demonstrated a gene signature of DCM. Although proteomics revealed only mild changes in global protein expression in left ventricular tissue of mutant mice, a glycoproteomic analysis unveiled broad glycosylation changes with significant alterations in sarcolemmal proteins including different subunits of laminin-211, which was confirmed by immunoblot analyses. Finally, augmentation of PGM1 in KO mice via AAV9-PGM1 gene replacement therapy prevented and halted the progression of the DCM phenotype.

Activating P2Y1 receptors improves function in arteries with repressed autophagy.

AIM: The importance of endothelial cell (EC) autophagy to vascular homeostasis in the context of health and disease is evolving. Earlier, we reported that intact EC autophagy is requisite to maintain shear-stress-induced nitric oxide (NO) generation via glycolysis-dependent purinergic signalling to endothelial NO synthase (eNOS). Here, we illustrate the translational and functional significance of these findings. METHODS AND RESULTS: First, we assessed translational relevance using older male humans and mice that exhibit blunted EC autophagy and impaired arterial function vs. adult controls. Active hyperaemia evoked by rhythmic handgrip exercise-elevated radial artery shear-rate similarly from baseline in adult and older subjects for 60 min. Compared with baseline, indexes of autophagy initiation, p-eNOSS1177 activation, and NO generation, occurred in radial artery ECs obtained from adult but not older volunteers. Regarding mice, indexes of autophagy and p-eNOSS1177 activation were robust in ECs from adult but not older animals that completed 60-min treadmill-running. Furthermore, 20 dyne • cm2 laminar shear stress × 45-min increased autophagic flux, glycolysis, ATP production, and p-eNOSS1177 in primary arterial ECs obtained from adult but not older mice. Concerning functional relevance, we next questioned whether the inability to initiate EC autophagy, glycolysis, and p-eNOSS1177in vitro precipitates arterial dysfunction ex vivo. Compromised intraluminal flow-mediated vasodilation displayed by arteries from older vs. adult mice was recapitulated in vessels from adult mice by (i) NO synthase inhibition; (ii) acute autophagy impairment using 3-methyladenine (3-MA); (iii) EC Atg3 depletion (iecAtg3KO mice); (iv) purinergic 2Y1-receptor (P2Y1-R) blockade; and (v) germline depletion of P2Y1-Rs. Importantly, P2Y1-R activation using 2-methylthio-ADP (2-Me-ADP) improved vasodilatory capacity in arteries from (i) adult mice treated with 3-MA; (ii) adult iecAtg3KO mice; and (iii) older animals with repressed EC autophagy. CONCLUSIONS: Arterial dysfunction concurrent with pharmacological, genetic, and age-associated EC autophagy compromise is improved by activating P2Y1-Rs.

A Fluorogenic-Based Assay to Measure Chaperone-Mediated Autophagic Activity in Cells and Tissues.

Objective: Pathologies including cardiovascular diseases, cancer, and neurological disorders are caused by the accumulation of misfolded / damaged proteins. Intracellular protein degradation mechanisms play a critical role in the clearance of these disease-causing proteins. Chaperone mediated autophagy (CMA) is a protein degradation pathway that employs chaperones to bind proteins, bearing a unique KFERQ-like motif, for delivery to a CMA-specific Lysosome Associated Membrane Protein 2a (LAMP2a) receptor for lysosomal degradation. To date, steady-state CMA function has been assessed by measuring LAMP2A protein expression. However, this does not provide information regarding CMA degradation activity. To fill this dearth of tools / assays to measure CMA activity, we generated a CMA-specific fluorogenic substrate assay. Methods: A KFERQ-AMC [Lys-Phe-Asp-Arg-Gln-AMC(7-amino-4-methylcou-marin)] fluorogenic CMA substrate was synthesized from Solid-Phase Peptide Synthesis. KFERQ-AMC, when cleaved via lysosomal hydrolysis, causes AMC to release and fluoresce (Excitation:355 nm, Emission:460 nm). Using an inhibitor of lysosomal proteases, i.e., E64D [L-trans-Epoxy-succinyl-leucylamido(4-guanidino)butane)], responsible for cleaving CMA substrates, the actual CMA activity was determined. Essentially, CMA activity = (substrate) fluorescence - (substrate+E64D) fluorescence . To confirm specificity of the KFERQ sequence for CMA, negative control peptides were used. Results: Heart, liver, and kidney lysates containing intact lysosomes were obtained from 4-month-old adult male mice. First, lysates incubated with KFERQ-AMC displayed a time dependent (0-5 hour) increase in AMC fluorescence vs. lysates incubated with negative control peptides. These data validate the specificity of KFERQ for CMA. Of note, liver exhibited the highest CMA (6-fold; p<0.05) > kidney (2.4-fold) > heart (0.4-fold) at 5-hours. Second, E64D prevented KFERQ-AMC degradation, substantiating that KFERQ-AMC is degraded via lysosomes. Third, cleavage of KFERQ-AMC and resulting AMC fluorescence was inhibited in Human embryonic kidney (HEK) cells and H9c2 cardiac cells transfected with Lamp2a vs. control siRNA. Further, enhancing CMA using Lamp2a adenovirus upregulated KFERQ degradation. These data suggest that LAMP2A is required for KFERQ degradation. Conclusion. We have generated a novel assay for measuring CMA activity in cells and tissues in a variety of experimental contexts.

Reduce, Reuse, Recycle, Run ! : 4 Rs to improve cardiac health in advanced age.

During the aging process damaged/dysfunctional proteins and organelles accumulate and contribute to organ dysfunction. Luckily, there is a conserved intracellular process to reuse and recycle these dysregulated cellular components termed macroautophagy (autophagy). Unfortunately, strong evidence indicates autophagy is compromised with aging, protein quality control is jeopardized, and resultant proteotoxicity can contribute significantly to age-associated organ dysfunction. Are there interventions that can re-establish autophagic flux that is otherwise impaired with aging? With particular regard to the heart, here we review evidence that caloric-restriction, the polyamine spermidine, and the mTOR inhibitor rapamycin, even when initiated late-in-life, restore cardiomyocyte autophagy to an extent that lessens age-associated cardiac dysfunction. Cho et al. provide a physiological intervention to this list i.e., regular physical exercise initiated late-in-life boosts cardiomyocyte autophagic flux and rejuvenates cardiac function in male mice. While this study provides strong evidence for a mechanism whereby heightened physical activity can lead to improved heart health in the context of aging, (i) only male mice were studied; (ii) the intensity of exercise-training might not be suitable for all; and (iii) mice with aging-associated comorbidities were not investigated. Nonetheless, Cho et al. provide robust evidence that a low-cost and simple behavioral intervention initiated late-in-life improves cardiomyocyte autophagic flux and rejuvenates cardiac function.

Chaperone-mediated autophagy protects cardiomyocytes against hypoxic-cell death.

Chaperone-mediated autophagy (CMA) is a chaperone-dependent process of selective cytosolic protein turnover that targets specific proteins to lysosomes for degradation. Enhancing protein degradation mechanisms has been shown to be beneficial in multiple models of cardiac disease, including myocardial infarction (MI) and ischemia-reperfusion (I/R) injury. However, the causal role of CMA in cardiomyocyte injury and death is largely unknown. Hypoxia is an important contributor to both MI and I/R damage, which are major, precedent causes of heart failure. Upregulating CMA was hypothesized to protect against hypoxia-induced cardiomyocyte death. Lysosome-associated membrane protein 2a (Lamp2a) overexpression and knockdown were used to causally study CMA's role in hypoxically stressed cardiomyocytes. LAMP2a protein levels were used as both a primary indicator and driver of CMA function. Hypoxic stress was stimulated by CoCl2 treatment, which increased LAMP2a protein levels (+1.4-fold) and induced cardiomyocyte apoptosis (+3.2-4.0-fold). Lamp2a siRNA knockdown (-3.2-fold) of control cardiomyocytes increased apoptosis (+1.8-fold) suggesting that loss of CMA is detrimental for cardiomyocyte survival. However, there was neither an additive nor a synergistic effect on cell death when Lamp2a-silenced cells were treated with CoCl2. Conversely, Lamp2a overexpression (+3.0-fold) successfully reduced hypoxia-induced apoptosis by ∼50%. LAMP2a was also significantly increased (+1.7-fold) in ischemic heart failure patient samples, similar to hypoxically stressed cardiomyocytes. The failing ischemic hearts may have had insufficient CMA activation. To our knowledge, this study for the first time establishes a protective role for CMA (via Lamp2a overexpression) against hypoxia-induced cardiomyocyte loss and reveals the intriguing possibility that CMA activation may offer a cardioprotective treatment for ischemic heart disease.

The Effects of Exercise on White and Brown Adipose Tissue Cellularity, Metabolic Activity and Remodeling.

Emerging evidence suggests a significant functional role of adipose tissue in maintaining whole-body metabolic health. It is well established that obesity leads to compositional and morphological changes in adipose tissue that can contribute to the development of cardiometabolic disorders. Thus, the function and size of adipocytes as well as perfusion and inflammation can significantly impact health outcomes independent of body mass index. Lifestyle interventions such as exercise can improve metabolic homeostasis and reduce the risk for developing cardiometabolic disorders. Adipose tissue displays remarkable plasticity in response to external stimuli such as dietary intervention and exercise. Here we review systemic and local effects of exercise that modulate white and brown adipose tissue cellularity, metabolic function and remodeling in humans and animals.

Late-in-life treadmill training rejuvenates autophagy, protein aggregate clearance, and function in mouse hearts.

Protein quality control mechanisms decline during the process of cardiac aging. This enables the accumulation of protein aggregates and damaged organelles that contribute to age-associated cardiac dysfunction. Macroautophagy is the process by which post-mitotic cells such as cardiomyocytes clear defective proteins and organelles. We hypothesized that late-in-life exercise training improves autophagy, protein aggregate clearance, and function that is otherwise dysregulated in hearts from old vs. adult mice. As expected, 24-month-old male C57BL/6J mice (old) exhibited repressed autophagosome formation and protein aggregate accumulation in the heart, systolic and diastolic dysfunction, and reduced exercise capacity vs. 8-month-old (adult) mice (all p < 0.05). To investigate the influence of late-in-life exercise training, additional cohorts of 21-month-old mice did (old-ETR) or did not (old-SED) complete a 3-month progressive resistance treadmill running program. Body composition, exercise capacity, and soleus muscle citrate synthase activity improved in old-ETR vs. old-SED mice at 24 months (all p < 0.05). Importantly, protein expression of autophagy markers indicate trafficking of the autophagosome to the lysosome increased, protein aggregate clearance improved, and overall function was enhanced (all p < 0.05) in hearts from old-ETR vs. old-SED mice. These data provide the first evidence that a physiological intervention initiated late-in-life improves autophagic flux, protein aggregate clearance, and contractile performance in mouse hearts.

Protein and Mitochondria Quality Control Mechanisms and Cardiac Aging.

Cardiovascular disease (CVD) is the number one cause of death in the United States. Advancing age is a primary risk factor for developing CVD. Estimates indicate that 20% of the US population will be ≥65 years old by 2030. Direct expenditures for treating CVD in the older population combined with indirect costs, secondary to lost wages, are predicted to reach $1.1 trillion by 2035. Therefore, there is an eminent need to discover novel therapeutic targets and identify new interventions to delay, lessen the severity, or prevent cardiovascular complications associated with advanced age. Protein and organelle quality control pathways including autophagy/lysosomal and the ubiquitin-proteasome systems, are emerging contributors of age-associated myocardial dysfunction. In general, two findings have sparked this interest. First, strong evidence indicates that cardiac protein degradation pathways are altered in the heart with aging. Second, it is well accepted that damaged and misfolded protein aggregates and dysfunctional mitochondria accumulate in the heart with age. In this review, we will: (i) define the different protein and mitochondria quality control mechanisms in the heart; (ii) provide evidence that each quality control pathway becomes dysfunctional during cardiac aging; and (iii) discuss current advances in targeting these pathways to maintain cardiac function with age.

Unlocking the Secrets of Mitochondria in the Cardiovascular System: Path to a Cure in Heart Failure­A Report from the 2018 National Heart, Lung, and Blood Institute Workshop

Mitochondria have emerged as a central factor in the pathogenesis and progression of heart failure, and other cardiovascular diseases, as well, but no therapies are available to treat mitochondrial dysfunction. The National Heart, Lung, and Blood Institute convened a group of leading experts in heart failure, cardiovascular diseases, and mitochondria research in August 2018. These experts reviewed the current state of science and identified key gaps and opportunities in basic, translational, and clinical research focusing on the potential of mitochondria-based therapeutic strategies in heart failure. The workshop provided short- and long-term recommendations for moving the field toward clinical strategies for the prevention and treatment of heart failure and cardiovascular diseases by using mitochondria-based approaches.

Identification of a Paracrine Signaling Mechanism Linking CD34high Progenitors to the Regulation of Visceral Fat Expansion and Remodeling.

Accumulation of visceral (VIS) is a predictor of metabolic disorders and insulin resistance. This is due in part to the limited capacity of VIS fat to buffer lipids allowing them to deposit in insulin-sensitive tissues. Mechanisms underlying selective hypertrophic growth and tissue remodeling properties of VIS fat are not well understood. We identified subsets of adipose progenitors (APs) unique to VIS fat with differential Cd34 expression and adipogenic capacity. VIS low (Cd34 low) APs are adipogenic, whereas VIS high (Cd34 high) APs are not. Furthermore, VIS high APs inhibit adipogenic differentiation of SUB and VIS low APs in vitro through the secretion of soluble inhibitory factor(s). The number of VIS high APs increased with adipose tissue expansion, and their abundance in vivo caused hypertrophic growth, fibrosis, inflammation, and metabolic dysfunction. This study unveils the presence of APs unique to VIS fat involved in the paracrine regulation of adipogenesis and tissue remodeling.