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Nicotinamide adenine dinucleotide (NAD

We examined the effects of SARM1 deletion on NAD+ pool, transcripts of NAD+ metabolism, and fibrotic pathway for the first time in mouse hearts. We observed the sexually dimorphic effects of SARM1 deletion. How these sex-dependent effects influence the outcomes of SARM1 deficiency in male and female mice in responses to cardiac stresses warrant further investigation. The elevation of cardiac NAD+ pool by SARM1 deletion provides evidence that targeting SARM1 may reverse disease-related NAD+ decline.

The pathology of aging impacts multiple organ systems including the kidney, skeletal, and cardiac muscle. Long-term treatment with the mitochondrial targeted peptide elamipretide has previously been shown to improvein vivomitochondrial function in aged mice that is associated with increased fatigue resistance and treadmill performance, improved cardiovascular diastolic function, and glomerular architecture of the kidney. However, elamipretide is a short tetrameric peptide that is not orally bioavailable limiting its routes of administration. This study tested whether twice weekly intermittent injections of elamipretide could recapitulate the same functional improvements as continuous long-term infusion. We found that intermittent treatment with elamipretide for 8 months preserved endurance running in mice, skeletal muscle force production, and left ventricular mass but did not affect heart or kidney function as previously reported using continuous treatment.

Cardiac arrhythmia is triggered by deregulated ion channels and abnormal electrical activity, and it increases susceptibility of patients to sudden death. The regulations of ion movement in and out of the myocytes or subcellular compartments are highly energy-dependent. Although impaired energetics has been implicated in arrhythmia, the causal roles and intricate details of how mitochondrial dysfunction contributes to arrhythmogenesis are far from established. We employed a mouse model with cardiac-specific mitochondrial dysfunction by deleting Ndufs4, a protein of mitochondrial complex I (Ndufs4-cKO). Complex I deficiency in Ndufs4-cKO mice lowered cardiac NAD/NADH ratio but did not affect cardiac function and geometry at baseline. Control and Ndufs4-cKO mice showed normal sinus rhythm at baseline. Ndufs4-cKO mice had 3-fold increases non-sinus arrhythmic events upon an arrhythmogenic stress test. 50% of Ndufs4-cKO mice succumbed to sudden cardiac death with displays of pre-ventricular contractions and ventricular fibrillation. We collected cardiac tissues at baseline and post stress. We observed increased ryanodine receptor, RyR2-S2814 phosphorylation, reduced phospholamban PLN S16 phosphorylation and decreased calsequestrin 2 expression in Ndufs4-cKO hearts post stress, but not at baseline. Isolated Ndfus4-cKO cardiomyocytes after stress test show a trend towards prolonged time to peak shortening and time to peak Ca 2+ transient. In addition, elevating cardiac NAD levels lowered arrhythmic events of Ndufs4-cKO mice after stress test. These results revealed direct evidence that mitochondrial dysfunction increases susceptibility of hearts to arrhythmia by regulating NAD redox balance and SR calcium homeostasis.Ndufs4-cKO mice with chronic high fat diet (HFD) had accelerated declines in systolic and diastolic function and increased hypertrophy, compared to control mice. WT mice with HFD showed diastolic dysfunction and increased arrhythmic events after stress test, while diastolic dysfunction was reversed by elevating cardiac NAD levels. Our data suggest an arrhythmogenic mechanism linking mitochondrial dysfunction and NAD redox imbalance with SR calcium homeostasis, which may explain HFD-induced arrhythmogenesis.

This review summarizes current understanding on the roles of nicotinamide adenine dinucleotide (NADNAD

BACKGROUND: Diabetes is a risk factor of heart failure and promotes cardiac dysfunction. Diabetic tissues are associated with NAD(+) redox imbalance; however, the hypothesis that NAD(+) redox imbalance causes diabetic cardiomyopathy (DCM) has not been tested. This investigation employed mouse models with altered NAD(+) redox balance to test this hypothesis. METHODS: Diabetic stress was induced in mice by streptozotocin. Cardiac function was measured by echocardiography. Heart and plasma samples were collected for biochemical, histological and molecular analyses. Two mouse models with altered NAD(+) redox states (cardiac-specific knockout mice of Ndufs4, cKO; nicotinamide phosphoribosyltransferase transgenic mice, NAMPT) were used. RESULTS: Diabetic stress caused cardiac dysfunction and lowered NAD(+)/NADH in wild-type mice. Mice with lowered cardiac NAD(+)/NADH without baseline dysfunction, cKO mice, were challenged with chronic diabetic stress. NAD(+) redox imbalance in cKO hearts exacerbated systolic (FS: 27.6% vs 36.9% at 4-week, male cohort, P

Background: Diabetes is a risk factor for heart failure and promotes cardiac dysfunction. Diabetic tissues are associated with nicotinamide adenine dinucleotide (NAD + ) redox imbalance; however, the hypothesis that NAD + redox imbalance causes diabetic cardiomyopathy has not been tested. This investigation used mouse models with altered NAD + redox balance to test this hypothesis. Methods: Diabetic stress was induced in mice by streptozotocin. Cardiac function was measured by echocardiography. Heart and plasma samples were collected for biochemical, histological, and molecular analyses. Two mouse models with altered NAD + redox states (1, Ndufs4 [NADH:ubiquinone oxidoreductase subunit S4] knockout, cKO, and 2, NAMPT [nicotinamide phosphoribosyltranferase] transgenic mice, NMAPT) were used. Results: Diabetic stress caused cardiac dysfunction and lowered NAD + /NADH ratio (oxidized/reduced ratio of nicotinamide adenine dinucleotide) in wild-type mice. Mice with lowered cardiac NAD + /NADH ratio without baseline dysfunction, cKO mice, were challenged with chronic diabetic stress. NAD + redox imbalance in cKO hearts exacerbated systolic (fractional shortening: 27.6% versus 36.9% at 4 weeks, male cohort P P + redox imbalance promoted superoxide dismutase 2 acetylation, protein oxidation, troponin I S150 phosphorylation, and impaired energetics in diabetic cKO hearts. Importantly, elevation of cardiac NAD + levels by NAMPT normalized NAD + redox balance, alleviated cardiac dysfunction (fractional shortening: 40.2% versus 24.8% in cKO:NAMPT versus cKO, P P Conclusions: Our results show that NAD + redox imbalance to regulate acetylation and phosphorylation is a critical mediator of the progression of diabetic cardiomyopathy and suggest the therapeutic potential for diabetic cardiomyopathy by harnessing NAD + metabolism.

Free radicals, or reactive oxygen species, have been implicated as one of the primary causes of myocardial pathologies elicited by chronic diseases and age. The imbalance between pro-oxidants and antioxidants, termed “oxidative stress”, involves several pathological changes in mouse hearts, including hypertrophy and cardiac dysfunction. However, the molecular mechanisms and adaptations of the hearts in mice lacking cytoplasmic superoxide dismutase (Sod1KO) have not been investigated. We used echocardiography to characterize cardiac function and morphology in vivo. Protein expression and enzyme activity of Sod1KO were confirmed by targeted mass spectrometry and activity gel. The heart weights of the Sod1KO mice were significantly increased compared with their wildtype peers. The increase in heart weights was accompanied by concentric hypertrophy, posterior wall thickness of the left ventricles (LV), and reduced LV volume. Activated downstream pathways in Sod1KO hearts included serine–threonine kinase and ribosomal protein synthesis. Notably, the reduction in LV volume was compensated by enhanced systolic function, measured by increased ejection fraction and fractional shortening. A regulatory sarcomeric protein, troponin I, was hyper-phosphorylated in Sod1KO, while the vinculin protein was upregulated. In summary, mice lacking cytoplasmic superoxide dismutase were associated with an increase in heart weights and concentric hypertrophy, exhibiting a pathological adaptation of the hearts to oxidative stress.

Diabetes is a risk factor of heart failure and leads to cardiac dysfunction, a condition named diabetic cardiomyopathy (DCM). NAD redox imbalance is observed in diabetic tissues. However, whether NAD redox imbalance promotes cardiac dysfunction in DCM remains unknown. The objective of this study is to determine the causal role of NAD redox imbalance in DCM.Type 1 diabetes was induced in C57BL6 mice by streptozotocin (STZ) injections, and DCM was allowed to develop for 16 weeks. Diabetes-induced chronic stress led to systolic and diastolic cardiac dysfunction along with a lowered NAD/NADH ratio. The diabetogenic protocol was applied to control and cardiac-specific Ndufs4-KO mice (cKO), a mouse model with lowered cardiac NAD/NADH without baseline cardiac dysfunction. Analyses of blood glucose and >200 metabolites in plasma showed no significant change in diabetic control and diabetic cKO mice, suggesting that their hearts experienced similar diabetic stress. Diabetic cKO hearts showed lowered NAD/NADH, aggravated contractile and relaxation dysfunction compared to the diabetic control hearts in both sexes. The data suggest that NAD redox imbalance exacerbated DCM. Collagen staining and transcript analyses of fibrosis-related genes showed no change in diabetic cKO hearts, signifying that the exacerbated dysfunction was due to cardiomyocyte dysfunction. A global protein acetylation was promoted in the diabetic cKO hearts with an increase in SOD2 acetylation levels at lysine-68 (SOD2-K68Ac) and enhanced protein oxidation. Diabetic cKO mice displayed enhanced levels of TnI S150 phosphorylation (TnI-S150Pi), but not phosphorylation of TnI-S23/24 or MyBPc-S282. These data suggest that enhanced oxidative stress and altered myofilament Ca 2+ sensitivity via TnI-S150Pi are responsible for the NAD redox imbalance-exacerbated DCM.To confirm the role of NAD redox imbalance in DCM, we elevated cardiac NAD levels in diabetic cKO mice by cardiac-specific expression of NAMPT. Expression of NAMPT in the heart improved cardiac systolic and diastolic function in diabetic cKO hearts. This was due to increased NAD/NADH ratio and reversed pathogenic mechanisms (lowered SOD2-K68Ac and TnI-S150Pi). This study supports the causal role of NAD redox imbalance in DCM.

Diabetes is long linked to lowered NAD/NADH ratio, aka NAD redox imbalance, but its causal role to diabetic cardiomyopathy is not established. We used mouse models with latent decrease in cardiac NAD/NADH ratio (cardiac-specific Ndufs4-KO, cKO) and elevated cardiac NAD levels to directly test whether cardiac NAD redox imbalance accelerates diabetic cardiomyopathy. Control and cKO mice were subjected to 8-week T1D stress, and longitudinal cardiac function was measured by echocardiography. Accelerated declines in systolic and diastolic function were observed in T1D cKO mice. Insulin depletion and hyperglycemia were similar in T1D control and T1D cKO mice, and serum metabolomic analyses showed unchanged aqueous and lipid metabolite levels. These metabolite results suggested that T1D control and cKO hearts were stressed under similar diabetic conditions. Importantly, elevation of cardiac NAD levels to attenuate NAD redox imbalance mitigated the accelerated functional declines in T1D cKO hearts. The data from mouse models with manipulated NAD redox states suggested that NAD redox imbalance accelerates diabetic cardiomyopathy. Cardiac fibrosis levels were not different in T1D control and cKO hearts, while transcript levels of fibrotic genes, including Adamts proteinases, integrins, laminins, matrix metalloproteinases and collagens, also showed no difference. Therefore, the accelerated functional declines in T1D cKO hearts are not due to altered extracellular matrix environment, but are rather due to cardiomyocyte dysfunction. We next determined whether the accelerated cardiac dysfunction is mediated via protein acetylation and oxidative stress. NAD-dependent global protein acetylation and inhibitory acetylation of superoxide dismutase 2 were elevated in T1D cKO hearts. Inhibition of SOD2 concomitantly promoted elevation of protein oxidation levels in T1D cKO hearts. The results suggested that NAD redox balance-dependent protein acetylation regulates oxidative stress to promote diabetic cardiomyopathy.

The generation of big data has enabled systems-level dissections into the mechanisms of cardiovascular pathology. Integration of genetic, proteomic, and pathophysiological variables across platforms and laboratories fosters discoveries through multidisciplinary investigations and minimizes unnecessary redundancy in research efforts. The Mouse Heart Attack Research Tool (mHART) consolidates a large data set of over 10 yr of experiments from a single laboratory for cardiovascular investigators to generate novel hypotheses and identify new predictive markers of progressive left ventricular remodeling after myocardial infarction (MI) in mice. We designed the mHART REDCap database using our own data to integrate cardiovascular community participation. We generated physiological, biochemical, cellular, and proteomic outputs from plasma and left ventricles obtained from post-MI and no-MI (naïve) control groups. We included both male and female mice ranging in age from 3 to 36 mo old. After variable collection, data underwent quality assessment for data curation (e.g., eliminate technical errors, check for completeness, remove duplicates, and define terms). Currently, mHART 1.0 contains >888,000 data points and includes results from >2,100 unique mice. Database performance was tested, and an example is provided to illustrate database utility. This report explains how the first version of the mHART database was established and provides researchers with a standard framework to aid in the integration of their data into our database or in the development of a similar database. NEW & NOTEWORTHY The Mouse Heart Attack Research Tool combines >888,000 cardiovascular data points from >2,100 mice. We provide this large data set as a REDCap database to generate novel hypotheses and identify new predictive markers of adverse left ventricular remodeling following myocardial infarction in mice and provide examples of use. The Mouse Heart Attack Research Tool is the first database of this size that integrates data sets across platforms that include genomic, proteomic, histological, and physiological data.

Although age-associated changes in kidney glomerular architecture have been described in mice and man, the mechanisms are unknown. It is unclear if these changes can be prevented or even reversed by systemic therapies administered at advanced age. Using light microscopy and transmission electron microscopy, our results showed glomerulosclerosis with injury to mitochondria in glomerular epithelial cells in mice aged 26 months (equivalent to a 79-year-old human). To test the hypothesis that reducing mitochondrial damage in late age would result in lowered glomerulosclerosis, we administered the mitochondrial targeted peptide, SS-31, to aged mice. Baseline (24-month-old) mice were randomized to receive 8 weeks of SS-31, or saline, and killed at 26 months of age. SS-31 treatment improved age-related mitochondrial morphology and glomerulosclerosis. Assessment of glomeruli revealed that SS-31 reduced senescence (p16, senescence-associated-s-Gal) and increased the density of parietal epithelial cells. However, SS-31 treatment reduced markers of parietal epithelial cell activation (Collagen IV, pERK1/2, and α - smooth muscle actin). SS-31 did not impact podocyte density, but it reduced markers of podocyte injury (desmin) and improved cytoskeletal integrity (synaptopodin). This was accompanied by higher glomerular endothelial cell density (CD31). Thus, despite initiating therapy in late-age mice, a short course of SS-31 has protective benefits on glomerular mitochondria, accompanied by temporal changes to the glomerular architecture. This systemic pharmacological intervention in old-aged animals limits glomerulosclerosis and senescence, reduces parietal epithelial cell activation, and improves podocyte and endothelial cell integrity.

Even in healthy aging, cardiac morbidity and mortality increase with age in both mice and humans. These effects include a decline in diastolic function, left ventricular hypertrophy, metabolic substrate shifts, and alterations in the cardiac proteome. Previous work from our laboratory indicated that short‐term (10‐week) treatment with rapamycin, an mTORC1 inhibitor, improved measures of these age‐related changes. In this report, we demonstrate that the rapamycin‐dependent improvement of diastolic function is highly persistent, while decreases in both cardiac hypertrophy and passive stiffness are substantially persistent 8 weeks after cessation of an 8‐week treatment of rapamycin in both male and female 22‐ to 24‐month‐old C57BL/6NIA mice. The proteomic and metabolomic abundance changes that occur after 8 weeks of rapamycin treatment have varying persistence after 8 further weeks without the drug. However, rapamycin did lead to a persistent increase in abundance of electron transport chain (ETC) complex components, most of which belonged to Complex I. Although ETC protein abundance and Complex I activity were each differentially affected in males and females, the ratio of Complex I activity to Complex I protein abundance was equally and persistently reduced after rapamycin treatment in both sexes. Thus, rapamycin treatment in the aged mice persistently improved diastolic function and myocardial stiffness, persistently altered the cardiac proteome in the absence of persistent metabolic changes, and led to persistent alterations in mitochondrial respiratory chain activity. These observations suggest that an optimal translational regimen for rapamycin therapy that promotes enhancement of healthspan may involve intermittent short‐term treatments., Cardiac morbidity and mortality increase with age in both mice and humans. Here, we demonstrate that 8‐week treatment with rapamycin is sufficient to persistently improve diastolic function, decrease age‐related cardiac hypertrophy and passive stiffness, and modulate the cardiac proteome, even a further 8 weeks after cessation of treatment. These observations suggest that an optimal translational regimen for rapamycin therapy that promotes enhancement of healthspan may involve intermittent short‐term treatments.

Inhibition of mammalian target of rapamycin complex I (mTORC1) by rapamycin improves cardiac function in both aging and heart failure. While the protective mechanisms are not fully understood in mammals, they are presumably mediated through metabolic regulation and suppression of protein translation by reduced phosphorylation of 4EBP1, a target of mTORC1. Using transverse aortic constriction (TAC) and Gαq overexpression-induced heart failure models, we examined the effect of cardiac-specific heterozygous deletion (het) of Raptor, a component of mTORC1, and cardiac-specific transgenic overexpression of wild type or phosphorylation site mutant 4EBP1. In wild-type mice with TAC-induced heart failure, quantitative shotgun proteomics revealed decreased abundance of proteins of mitochondrial metabolism and increased abundance of proteins in oxidative stress response, ubiquitin, and other pathways. The Raptor het ameliorated both TAC- and Gαq overexpression-induced heart failure and the associated proteomic remodeling, especially those pathways involved in mitochondrial function, citric acid cycle, and ubiquitination. In contrast, transgenic overexpression of either wild type or mutant 4EBP1 aggravated TAC and Gαq, consistent with reduced adaptive hypertrophy by suppression of protein translation, in parallel with adverse remodeling of left ventricular proteomes. Partial mTORC1 inhibition by Raptor heterozygous deletion ameliorates heart failure and is associated with better preservation of the mitochondrial proteome; however, this effect does not appear to be mediated through suppression of protein translation by increased 4EBP1. Increased activity of 4EBP1 reduced adaptive hypertrophy and aggravated heart failure, suggesting that protein translation is essential for adaptive hypertrophy in pressure overload. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s11357-019-00119-6) contains supplementary material, which is available to authorized users.

Aging is associated with a decline in diastolic function and is a strong risk factor for heart failure with preserved ejection fraction (HFpEF), which has no effective treatment. We previously showed that late-life rapamycin treatment can reverse age-related cardiac dysfunction in mice. However, the mechanisms of the reversal of diastolic dysfunction have not been established. The objective of this study is to determine the mechanisms by which rapamycin reverses age-related diastolic dysfunction. To study the effects of rapamycin on myocardial stiffness, we assessed the passive length-tension relationship of demembranated trabecular muscle from young, old control and old rapamycin-treated mice. We observed a substantial increase in the slope of the length-tension curve with aging, indicating an age-related increase in myocardial stiffness. The age-related increase in myocardial stiffness was significantly reduced by rapamycin treatment, by a mechanism independent of titin isoform shift. We measured the force-calcium relationship of the demembranated trabeculae and revealed an age-related increase in Ca 2+ sensitivity, as indicated by a left-shift of the force-pCa curve, which was partially restored after rapamycin treatment (pCa50: 5.59±0.04 for young controls; 5.76±0.04 for old controls; and 5.65±0.04 for old rapamycin-treated group). To investigate the changes at myofibril level, we assessed kinetic properties of control and rapamycin-treated myofibrils following maximal (pCa 4.0) and submaximal (pCa 5.6) Ca 2+ activation. Myofibrils from rapamycin-treated mice displayed increased rate of the fast phase of relaxation (kREL, fast) compared to old control at both maximal and submaximal Ca 2+ levels, potentially due to reduced myofibril Ca 2+ sensitivity. Studies have detected age-related reductions in expression and activity of SERCA2, which is responsible for SR calcium reuptake during diastole. In this study, we showed that rapamycin increased SERCA2 expression, which may improve cardiomyocyte relaxation. In summary, our results suggest that rapamycin normalizes age-related increases in myocardial stiffness and Ca 2+ sensitivity and improves myofibril relaxation kinetics, therefore, improving diastolic function.

Diabetes is linked to NAD(H) redox imbalance and lowered NAD + levels, but the roles of NAD + metabolism in diabetic cardiomyopathy are not established. We induced diabetes in C57BL6 wild type mice (WT) by streptozotocin (STZ) injections to cause depletion in serum insulin levels and elevation in fasting glucose levels. Hyperglycemic, male WT mice showed gradual declines in systolic and diastolic functions (FS, 50% vs 30%; E’/A’ ratio, 1.5 vs 1.0; 16-week after STZ), without causing cardiac hypertrophy. Dysfunctions in diabetic hearts were associated with lowered NAD + /NADH ratio and protein hyperacetylation, suggesting the roles of NAD(H) redox imbalance in diabetic cardiomyopathy. To test if NAD(H) redox imbalance promotes dysfunctions of diabetic hearts, we employed cardiac-specific Ndufs4-KO mice (cKO), which exhibit lowered NAD + /NADH ratio without overt cardiac dysfunction at baseline. Changes in cardiac functions at 2-, 4-, and 8-week after STZ injections, insulin and fasting glucose levels of control and cKO male mice were measured. Similar insulin depletion and hyperglycemia (~450 mg/dL) were observed in diabetic control and cKO mice. Systolic and diastolic dysfunctions were worsened in diabetic cKO mice (control vs cKO: FS 37% vs 22%; E’/A’ 1.25 vs 0.95, 8-week after STZ). Cardiac fibrosis levels were slightly elevated in diabetic control and cKO hearts, but were not different between the two groups (~4% in both group of mice). The results suggested that the accelerated declines in cardiac functions contributed by NAD(H) redox imbalance are not due to the altered extracellular matrix environment. To restore the NAD(H) redox balance in diabetic hearts, we elevated NAD + levels in male control and cKO mice with cardiac-specific NAMPT overexpression. Elevations in cardiac NAD + levels alleviated systolic and diastolic dysfunctions in diabetic control and cKO mice, despite similar levels of hyperglycemia among groups. Similar exacerbation of cardiac dysfunction was observed in female diabetic cKO mice, while NAMPT overexpression similarly improved cardiac function in female diabetic control and cKO mice. Our findings showed that NAD(H) redox imbalance promotes the progression of diabetic cardiomyopathy, via a fibrosis-independent mechanism.

Rapamycin, an inhibitor of mTOR signaling, has been shown to reverse diastolic dysfunction in old mice in 10 weeks, highlighting its therapeutic potential for a poorly treatable condition. However, the mechanisms and temporal regulation of its cardiac benefits remain unclear. We show that improved diastolic function in old mice begins at 2-4 weeks, progressing over the course of 10-week treatment. While TORC1-mediated S6 phosphorylation and TORC2 mediated AKT and PKCα phosphorylation are inhibited throughout the course of treatment, rapamycin inhibits ULK phosphorylation and induces autophagy during just the first week of treatment, returning to baseline at two weeks and after. Concordantly, markers of mitochondrial biogenesis increase over the first two weeks of treatment and return to control levels thereafter. This transient induction of autophagy and mitochondrial biogenesis suggests that damaged mitochondria are replaced by newly synthesized ones to rejuvenate mitochondrial homeostasis. This remodeling is shown to rapidly reverse the age-related reduction in fatty acid oxidation to restore a more youthful substrate utilization and energetic profile in old isolated perfused hearts, and modulates the myocardial metabolome in vivo. This study demonstrates the differential and dynamic mechanisms following rapamycin treatment and highlights the importance of understanding the temporal regulation of rapamycin effects.

Matrix metalloproteinases (MMPs) collectively degrade all extracellular matrix (ECM) proteins. Of the MMPs, MMP-9 has the strongest link to the development of cardiac dysfunction. Aging associates with increased MMP-9 expression in the left ventricle (LV) and reduced cardiac function. We investigated the effect of MMP-9 deletion on the cardiac ECM in aged animals.We used male and female middle-aged (10- to16-month old) and old (20- to 24-month old) wild-type (WT) and MMP-9 null mice (n = 6/genotype/age). LVs were decellularized to remove highly abundant mitochondrial proteins that could mask identification of relative lower abundant components, analyzed by shotgun proteomics, and proteins of interest validated by immunoblot.Elastin microfibril interface-located protein 1 (EMILIN-1) decreased with age in WT (p0.05), but not in MMP-9 null. EMILIN-1 promotes integrin-dependent cell adhesion and EMILIN-1 deficiency has been associated with vascular stiffening. Talin-2, a cytoskeletal protein, was elevated with age in WT (p0.05), and MMP-9 deficiency blunted this increase. Talin-2 is highly expressed in adult cardiac myocytes, transduces mechanical force to the ECM, and is activated by increases in substrate stiffness. Our results suggest that MMP-9 deletion may reduce age-related myocardial stiffness, which may explain improved cardiac function in MMP-9 null animals.We identified age-related changes in the cardiac proteome that are MMP-9 dependent, suggesting MMP-9 as a possible therapeutic target for the aging patient.

Half of the heart failure (HF) patients in US have preserved ejection fraction but diastolic dysfunction. Without a specific medication, better understanding on the mechanisms of diastolic dysfunction is urgently needed. NAD + -sensitive lysine acetylation emerges as regulators of HF pathogenesis. We generated mouse models to manipulate NAD + redox states in the heart. We used cardiac Ndufs4-KO mice (cKO) as a model of reduced NAD + /NADH ratio and cardiac NAMPT over expression (cNAMPT), as a model of elevated NAD + levels. Using labelled-free quantitative proteomics, we identified NAD + -sensitive changes of acetylation landscapes in the heart. Muscle contraction is one of the top categories of acetylated proteins identified by gene ontology analysis. Analysis of non-mitochondrial acetylome showed that many proteins regulating SR Ca 2+ homeostasis and myofilament movement were hyperacetylated in cKO hearts. Therefore, we hypothesized that increased acetylation of components of SR Ca 2+ homeostasis and myofilament movement impairs diastolic function. Using Doppler echocardiography, we observed a decline in E’/A’ ratio and increased myocardial performance index in cKO hearts. Systolic function and cardiac geometry were normal in cKO hearts. In addition, elevation NAD + levels by cNAMPT expression (cKO:cNAMPT) normalized protein hyperacetylation and reversed diastolic dysfunction in cKO. These data support that increased acetylation contributes to the decline in diastolic function in cKO hearts. Future experiments using isolated myofibril and cardiomyocytes will determine the roles of protein acetylation to regulate myofilament, SR Ca 2+ homeostasis and thus diastolic dysfunction.

The naked mole-rat (NMR) is a strictly subterranean rodent with a low resting metabolic rate. Nevertheless, it can greatly increase its metabolic activity to meet the high energetic demands associated with digging through compacted soils in its xeric natural habitat where food is patchily distributed. We hypothesized that the NMR heart would naturally have low basal function and exhibit a large cardiac reserve, thereby mirroring the species' low basal metabolism and large metabolic scope. Echocardiography showed that young (2–4 yr old) healthy NMRs have low fractional shortening (28 ± 2%), ejection fraction (43 ± 2%), and cardiac output (6.5 ± 0.4 ml/min), indicating low basal cardiac function. Histology revealed large NMR cardiomyocyte cross-sectional area (216 ± 10 μm2) and cardiac collagen deposition of 2.2 ± 0.4%. Neither of these histomorphometric traits was considered pathological, since biaxial tensile testing showed no increase in passive ventricular stiffness. NMR cardiomyocyte fibers showed a low degree of rotation, contributing to the observed low NMR cardiac contractility. Interestingly, when the exercise mimetic dobutamine (3 μg/g ip) was administered, NMRs showed pronounced increases in fractional shortening, ejection fraction, cardiac output, and stroke volume, indicating an increased cardiac reserve. The relatively low basal cardiac function and enhanced cardiac reserve of NMRs are likely to be ecophysiological adaptations to life in an energetically taxing environment.

The year 2013 has witnessed exciting advances in cardiovascular research exemplified by discovery leveraging genomics, proteomics and metabolomics platforms, genome editing, translation in individualized disease modeling, and application of regenerative therapies. New knowledge generated through integrative analysis has informed the understanding of molecular mechanisms that can now be validated and exploited to expedite management of biological processes. The Functional Genomics and Translational Biology Council from the American Heart Association (www.my.americanheart.org/fgtbcouncil) has the mission of advancing new discoveries in the fields of genetics, omics technologies, and translational biology and facilitate their application in cardiovascular health and disease. By creating a multidisciplinary collaborative environment, this Council integrates scientific knowledge from molecules to populations to contribute to the global American Heart Association goal of building healthier lives, free of cardiovascular diseases and stroke. With input from the Early Career Committee of the Council on Functional Genomics and Translational Biology, we considered many outstanding articles and selected 10 advances published during 2013. In this Special Report, we summarize some relevant articles related to each of those advances and highlight their significance in the cardiovascular field. Cardiac hypertrophy is an increasingly prevalent condition because of population aging with limited therapeutic options to prevent its progression to heart failure. Loffredo et al1 hypothesized that loss of systemic factors may be responsible for the stiffening and enlargement of the ventricles and thereby a therapeutic target for the treatment of systolic heart failure. Using a model of parabiosis (surgically linking the systemic circulation of 2 animals), they demonstrated that exposure to a young circulatory environment can reverse age-induced cardiac hypertrophy in old mice. Proteomic analysis of old and young adult plasma enabled identification of the growth differentiation factor 11, a member of the transforming growth factor-β superfamily, as a factor with decreasing blood levels in aging mice. …

The extracellular matrix (ECM) is a critical tissue component, providing structural support as well as important regulatory signaling cues to govern cellular growth, metabolism, and differentiation. The study of ECM proteins, however, is hampered by the low solubility of ECM components in common solubilizing reagents. ECM proteins are often not detected during proteomics analyses using unbiased approaches due to solubility issues and relatively low abundance compared to highly abundant cytoplasmic and mitochondrial proteins. Decellularization has become a common technique for ECM protein-enrichment and is frequently used in engineering studies. Solubilizing the ECM after decellularization for further proteomic examination has not been previously explored in depth. In this study, we describe testing of a series of protocols that enabled us to develop a novel optimized strategy for the enrichment and solubilization of ECM components. Following tissue decellularization, we use acid extraction and enzymatic deglycosylation to facilitate re-solubilization. The end result is the generation of three fractions for each sample: soluble components, cellular components, and an insoluble ECM fraction. These fractions, developed in mass spectrometry-compatible buffers, are amenable to proteomics analysis. The developed protocol allows identification (by mass spectrometry) and quantification (by mass spectrometry or immunoblotting) of ECM components in tissue samples. Biological significance The study of extracellular matrix (ECM) proteins in pathological and non-pathological conditions is often hampered by the low solubility of ECM components in common solubilizing reagents. Additionally, ECM proteins are often not detected during global proteomic analyses due to their relatively low abundance compared to highly abundant cytoplasmic and mitochondrial proteins. In this manuscript we describe testing of a series of protocols that enabled us to develop a final novel optimized strategy for the enrichment and solubilization of ECM components. The end result is the generation of three fractions for each sample: soluble components, cellular components, and an insoluble ECM fraction. By analysis of each independent fraction, differences in protein levels can be detected that in normal conditions would be masked. These fractions are amenable to mass spectrometry analysis to identify and quantify ECM components in tissue samples. The manuscript places a strong emphasis on the immediate practical relevance of the method, particularly when using mass spectrometry approaches; additionally, the optimized method was validated and compared to other methodologies described in the literature.

The free-radical theory of aging was proposed more than 50 years ago. As one of the most popular mechanisms explaining the aging process, it has been extensively studied in several model organisms. However, the results remain controversial. The mitochondrial version of free-radical theory of aging proposes that mitochondria are both the primary sources of reactive oxygen species (ROS) and the primary targets of ROS-induced damage. One critical ROS is hydrogen peroxide, which is naturally degraded by catalase in peroxisomes or glutathione peroxidase within mitochondria. Our laboratory developed mice-overexpressing catalase targeted to mitochondria (mCAT), peroxisomes (pCAT), or the nucleus (nCAT) in order to investigate the role of hydrogen peroxide in different subcellular compartments in aging and age-related diseases. The mCAT mice have demonstrated the largest effects on life span and healthspan extension. This chapter will discuss the mCAT phenotype and review studies using mCAT to investigate the roles of mitochondrial oxidative stresses in various disease models, including metabolic syndrome and atherosclerosis, cardiac aging, heart failure, skeletal muscle pathology, sensory defect, neurodegenerative diseases, and cancer. As ROS has been increasingly recognized as essential signaling molecules that may be beneficial in hormesis, stress response and immunity, the potential pleiotropic, or adverse effects of mCAT are also discussed. Finally, the development of small-molecule mitochondrial-targeted therapeutic approaches is reviewed.

Background: Impairments of mitochondrial function in the heart are linked intricately to the development of heart failure, but there is no therapy for mitochondrial dysfunction. Methods: We assessed the reduced/oxidized ratio of nicotinamide adenine dinucleotide (NADH/NAD + ratio) and protein acetylation in the failing heart. Proteome and acetylome analyses were followed by docking calculation, mutagenesis, and mitochondrial calcium uptake assays to determine the functional role of specific acetylation sites. The therapeutic effects of normalizing mitochondrial protein acetylation by expanding the NAD + pool also were tested. Results: Increased NADH/NAD + and protein hyperacetylation, previously observed in genetic models of defective mitochondrial function, also are present in human failing hearts as well as in mouse hearts with pathologic hypertrophy. Elevation of NAD + levels by stimulating the NAD + salvage pathway suppressed mitochondrial protein hyperacetylation and cardiac hypertrophy, and improved cardiac function in responses to stresses. Acetylome analysis identified a subpopulation of mitochondrial proteins that was sensitive to changes in the NADH/NAD + ratio. Hyperacetylation of mitochondrial malate-aspartate shuttle proteins impaired the transport and oxidation of cytosolic NADH in the mitochondria, resulting in altered cytosolic redox state and energy deficiency. Furthermore, acetylation of oligomycin-sensitive conferring protein at lysine-70 in adenosine triphosphate synthase complex promoted its interaction with cyclophilin D, and sensitized the opening of mitochondrial permeability transition pore. Both could be alleviated by normalizing the NAD + redox balance either genetically or pharmacologically. Conclusions: We show that mitochondrial protein hyperacetylation due to NAD + redox imbalance contributes to the pathologic remodeling of the heart via 2 distinct mechanisms. Our preclinical data demonstrate a clear benefit of normalizing NADH/NAD + imbalance in the failing hearts. These findings have a high translational potential as the pharmacologic strategy of increasing NAD + precursors are feasible in humans.

Aims Age-related diastolic dysfunction has been attributed to an increased passive stiffness, which is regulated by extracellular matrix (ECM). We recently showed that matrix metalloproteinase (MMP)-9, an ECM mediator, increases in the left ventricle (LV) with age. The aim of this study, accordingly, was to determine the role of MMP-9 in cardiac ageing. Methods and results We compared LV function in young (6–9 months), middle-aged (12–15 months), old (18–24 months) and senescent (26–34 months) wild-type (WT) and MMP-9 null mice ( n ≥ 12/group). All groups had similar fractional shortenings and aortic peak velocities, indicating that systolic function was not altered by ageing or MMP-9 deletion. The mitral ratios of early to late diastolic filling velocities were reduced in old and senescent WT compared with young controls, and this reduction was attenuated in MMP-9 null mice. Concomitantly, the increase in LV collagen content was reduced in MMP-9 null mice ( n = 5-6/group). To dissect the mechanisms of these changes, we evaluated the mRNA expression levels of 84 ECM and adhesion molecules by real-time qPCR ( n = 6/group). The expression of pro-fibrotic periostin and connective tissue growth factor (CTGF) increased with senescence, as did transforming growth factor-β (TGF-β)-induced protein levels and Smad signalling, and these increases were blunted by MMP-9 deletion. In senescence, MMP-9 deletion also resulted in a compensatory increase in MMP-8. Conclusion MMP-9 deletion attenuates the age-related decline in diastolic function, in part by reducing TGF-β signalling-induced periostin and CTGF expression and increasing MMP-8 expression to regulate myocardial collagen turnover and deposition.

Matrix metalloproteinase-7 (MMP-7) deletion has been shown to improve survival after myocardial infarction (MI). MMP-7 has a large array of in vitro substrates, but in vivo substrates for MMP-7 following MI have not been fully identified. Accordingly, we evaluated the infarct regions of wild-type (WT; n = 12) and MMP-7 null (null; n = 10) mice using a proteomic strategy. Seven days post-MI, infarct regions of the left ventricles were excised, homogenized, and protein extracts were analyzed by two-dimensional gel electrophoresis and mass spectrometry. Of 13 spots that showed intensity differences between WT and null, the intensities of eight spots were higher and those of five spots were lower in the null group (p < 0.05). Fibronectin and tenascin-C, known in vitro substrates of MMP-7, were identified in spots that showed lower intensity in the null. Immunoblotting and in vitro cleavage assays confirmed reduced fibronectin and tenascin-C fragment generation in the null, and this effect was restored by exogenous administration of MMP-7. Lower levels of full-length peroxiredoxin-1 and -2 and higher levels of the full-length peroxiredoxin-3 were detected in the null group, suggesting MMP-7 deletion may also indirectly regulate protein levels through nonenzymatic mechanisms. In conclusion, this is the first study to identify fibronectin and tenascin-C as in vivo MMP-7 substrates in the infarcted left ventricle using a proteomic approach.

Cardiovascular diseases are the leading cause of death for population over 65 years of age. With the progressive increase of population in this age group, it is crucial to understand the interaction between aging and cardiovascular diseases. Aging results in structural changes and functional decline of the cardiovascular system and is a major risk factor for cardiovascular diseases. Aging not only increases the prevalence of cardiovascular diseases but is also associated with impaired responses to cardiovascular diseases. This supports the notion that age-related changes in the cardiovascular system superimpose and interact with the pathophysiological mechanisms that lead to cardiovascular disease. This chapter will provide an overview of cardiovascular aging in humans and mammalian models, and discuss the recent advances in understanding how different hallmarks of aging play their roles in the pathogenesis of cardiovascular aging.

Cardiovascular diseases are the leading causes of death in the western hemisphere. The exponential increases in the incidence and mortality rate of cardiovascular diseases in the elderly suggest that age per se is a major risk factor for cardiovascular diseases. The Framingham Heart Study and the Baltimore Longitudinal Study on Aging demonstrated that in the elderly human population there is an age-dependent increase in left ventricular hypertrophy, diastolic dysfunction, and a decrease in exercise capacity. Many of these changes are closely recapitulated in animal models commonly used in aging study, including rodents, flies, and monkeys. The conservation of intrinsic cardiac aging in the Drosophila melanogaster model offers unique genetic insights into cardiac aging. The application of genetically modified aged rodents elucidates several critical molecular mechanisms involved in cardiac aging, including mitochondrial oxidative stress, nutrient signaling, neurohormonal regulation-insulin/IGF/PI3K pathway, and the adrenergic and renin–angiotensin–aldosterone systems. In addition, aging of cardiac stem cells and decreased cardiac functional reserve in aging contribute to the susceptibility of aged hearts to stress and disease. We further discuss the mechanism of progression to heart failure in aged hearts. As the projected number of elderly persons is predicted to double in the next 25 years and the prevalence and economic burden of age-related cardiovascular disabilities continues to increase, there is an urgent need to understand the biology of the aging heart, the mechanisms for age-dependent cardiac vulnerability and to use these insights to develop strategies to prevent heart failure in the elderly. Finally, the potential clinical application of several “anti-aging” strategies to treat cardiovascular diseases and to improve healthy cardiac aging is summarized, including calorie restriction and its mimetics, strategies of mitochondrial intervention, and other novel agents.

Aging results in progressive deteriorations in the structure and function of the heart and is a dominant risk factor for cardiovascular diseases, the leading cause of death in Western populations. Although the phenotypes of cardiac aging have been well characterized, the molecular mechanisms of cardiac aging are just beginning to be revealed. With the continuously growing elderly population, there is a great need for interventions in cardiac aging. This article will provide an overview of the phenotypic changes of cardiac aging, the molecular mechanisms underlying these changes, and will present some of the recent advances in the development of interventions to delay or reverse cardiac aging.

The mitochondrial respiratory chain (RC) produces most of the cellular ATP and requires strict quality-control mechanisms. To examine RC subunit proteostasis in vivo, we measured RC protein half-lives (HLs) in mice by liquid chromatography-tandem mass spectrometry with metabolic [2H3]-leucine heavy isotope labeling under divergent conditions. We studied 7 tissues/fractions of young and old mice on control diet or one of 2 diet regimens (caloric restriction or rapamycin) that altered protein turnover (42 conditions in total). We observed a 6.5-fold difference in mean HL across tissues and an 11.5-fold difference across all conditions. Normalization to the mean HL of each condition showed that relative HLs were conserved across conditions (Spearman’s ρ = 0.57; P < 10–4), but were highly heterogeneous between subunits, with a 7.3-fold mean range overall, and a 2.2- to 4.6-fold range within each complex. To identify factors regulating this conserved distribution, we performed statistical analyses to study the correlation of HLs to the properties of the subunits. HLs significantly correlated with localization within the mitochondria, evolutionary origin, location of protein-encoding, and ubiquitination levels. These findings challenge the notion that all subunits in a complex turnover at comparable rates and suggest that there are common rules governing the differential proteolysis of RC protein subunits under divergent cellular conditions.—Karunadharma, P. P., Basisty, N., Chiao, Y. A., Dai, D.-F., Drake, R., Levy, N., Koh, W. J., Emond, M. J., Kruse, S., Marcinek, D., Maccoss, M. J., Rabinovitch, P. S. Respiratory chain protein turnover rates in mice are highly heterogeneous but strikingly conserved across tissues, ages, and treatments.

Nitrogen (N) metabolism is essential for the biosynthesis of vital biomolecules. N status thus exerts profound effects on plant growth and development, and must be closely monitored. In bacteria and fungi, a few sophisticated N sensing systems have been extensively studied. In animals, the ability to receive amino acid signals has evolved to become an integral part of the nervous coordination system. In this review, we will summarize recent developments in the search for putative N sensing systems in higher plants based on homologous systems in bacteria, fungi, and animals. Apparently, although plants have separated and diversified from other organisms during the evolution process, striking similarities can be found in their N sensing systems compared with those of their counterparts; however, our understanding of these systems is still incomplete. Significant modifications of the N sensing systems (including cross-talk with other signal transduction pathways) in higher plants may be a strategy of adaptation to their unique mode of life. (Managing editor: Ping He)