CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62/216,698, filed September 10, 2015, U.S. Provisional Application No. 62/215,502, filed September 8, 2015, and U.S. Provisional Application No. 62/114,501, filed February 10, 2015, all of which are incorporated herein by reference in their entirety.

STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with government support under contract number R01 DK089705 awarded by the National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD

[0003] The present invention relates to methods for treating heart failure. BACKGROUND

[0004] Iron is required for oxygen transport, oxidative phosphorylation, DNA synthesis, and other cellular processes. Iron co-factors, for example, iron-sulfur (Fe-S) clusters and heme, are synthesized by mitochondria and needed for mitochondrial function. Mitochondria supply energy for cells, including the energy needed by cardiomyocytes for repeated heart muscle contraction. Accordingly, the maintenance of mitochondria, including the removal of dysfunctional mitochondria through mitophagy and synthesis of iron co-factors, is needed for cardiomyocyte function.

[0005] Improper cardiomyocyte function may lead to heart failure. Heart failure may be further complicated by the presence of iron deficiency. Up to 50% of patients with heart failure have iron deficiency, which is associated with poor outcomes despite current therapies. [0006] Accordingly, a need exists for the identification and development of new therapies for heart failure.

SUMMARY

[0007] The present invention relates to a method for treating heart failure in a subject in need thereof. The method may comprise administering a therapeutically effective amount of nicotinamide riboside to the subject.

[0008] The present invention also relates to a method for treating heart failure in a subj ect in need thereof. The method may comprise administering a therapeutically effective amount of nicotinamide riboside and a therapeutically effective amount of iron to the subject.

[0009] The present invention further relates to a method for treating heart failure associated with iron deficiency in a subject in need thereof. The method may comprise administering a therapeutically effective amount of nicotinamide riboside to the subject.

[0010] The present invention also relates to a method for treating heart failure associated with iron deficiency in a subject in need thereof. The method may comprise administering a therapeutically effective amount of nicotinamide riboside and a therapeutically effective amount of iron to the subject.

[0011] The present invention further relates to a method of identifying a subj ect suffering from heart failure as a candidate for treatment with nicotinamide riboside. The method may comprise (a) measuring in a sample obtained from the subject a level selected from the group consisting of: (i) a level of a ratio of NAD/NADH; (ii) a level of iron; (iii) a level of FGF21 protein; (iv) a level of Angiopoietin-like 4 (AngPtL4) protein; and (v) any combination thereof. The method may also comprise (b) comparing the measured level to a control level; and (c) identifying the subject as a candidate for treatment with nicotinamide riboside if (i) the measured level of the ratio of NAD/NADH is lower than the control level of the ratio of NAD/NADH; (ii) the measured level of iron is lower than the control level of iron; (iii) the measured level of FGF21 protein is higher than the control level of FGF21 protein; (iv) the measured level of AngPtL4 protein is higher than the control level of AngPtL4 protein; or (v) any combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 shows that loss of Tfrl in cardiomyocytes caused cardiomyopathy. (A)

Yfrihrt/hrt mice appeared similar to wild-type (WT) at P10. (B) H&E staining of heart sections P10 demonstrated cardiomegaly in xfr ¹ ^ ¹¹¹¹ mice. Scale bars = 1 mm. (C) xf _r i ^1111/1111 _m i _ce had increased heart to body weight ratios at P10. (D) Echocardiograms from representative xf _r i ^1111/1111 and WT littermates at P10. Upper panel, short axis; lower panel, long axis, xf _r i ^1111/1111 _m i _ce had markedly impaired cardiac function. (E) Left ventricular diameter and fractional shortening were abnormal in xf _r i ^1111/1111 mice. LVDd = left ventricular diameter in diastole; LVDs = left ventricular diameter in systole. (F) Representative images of WGA staining for cardiomyocyte morphometries and quantitation showing larger cardiomyocyte area in χβ-ι ^1111/1111 mice. Scale bars= 15 μιη. (G) mRNA levels of cardiac hypertrophy biomarkers as described in the Examples. Data were presented as means ± standard error of the mean (SEM). Sample size (n) was indicated. *p < 0.05; **p < 0.01; ***p < 0.001 by one-way ANOVA. See FIG. 8.

[0013] Figure 2 shows iron deficiency and insufficient Fe-S cluster biogenesis in xf _r i ^1111/1111 mice. (A-C) Non-heme iron levels in the heart at P5 (A) and P10 (B); total iron concentration at P10 to PI 1 (C). (D) Decreased Fe-S cluster proteins Dpyd and Ppat in hearts from xf _r i ^1111/1111 _m ice with Rpll9 control. Age and genotype were shown at the top. Data were presented as means ± SEM. Sample size (n) was indicated. ***p < 0.001 by one-way ANOVA.

[0014] Figure 3 shows abnormal mitochondrial morphology and function in hearts from

Tf _r ihrt/hrt mice. (A) Electron micrographs comparing mitochondria in WT and xf _r i ^1111/1111 hearts. _{Tfr l} hrt/h ⁱ t _mitoc hondria were slightly abnormal at P5, but markedly enlarged and disrupted at P10. Scale bars = 500 nm. (B) Representative protein levels for ETC complexes by immunoblot. (C) Enzymatic activity of complexes I to IV of ETC from P10 xf _r i ^1111/1111 _anc j γχ littermates. (D) Relative mRNA levels of Polrmt and mitochondria-encoded genes. (E) Relative mRNA levels of PGCl-a {Ppargcla) and PGCl-β {Ppargclb). (F) Relative mRNA levels of PPARa {Ppara), Rxr gamma {Rxrg), and fatty acid transport protein {Fatpl). Data were presented as means ± SEM. Sample size (n) was indicated

[0015] Figure 4 shows metabolic changes and increased apoptosis in hearts from Tfrl ^Mhrl mice at P10. (A) Relative mRNA levels of transcripts induced by hypoxia. (B) Relative mRNA levels of transcripts encoding enzymes of glycolysis. (C) Relative level of Myc mRNA. (D) TUNEL staining for apoptosis. Top row without DAPI staining of nuclei; bottom row with DAPI staining. Vertical pairs of panels from left to right: negative control, positive control, WT, and Yfrihrt/hrt. Bright green fluorescent nuclei represented apoptotic cells; scale bars = 100 μιη.

Results were quantified on the right; data were presented as means ± SEM. Sample size (n) was indicated. *p < 0.05; **p < 0.01; and ***p < 0.001 by one-way ANOVA.

[0016] Figure 5 shows altered expression of molecules involved in autophagy and mitophagy in hearts from Tfrl ^Mhrl mice. (A-G) Multiple autophagy- and mitophagy-related genes were examined in 10-day old heart samples for mRNA levels or, where feasible, for protein levels, as indicated in each panel. Differences indicated early induction of autophagy, but a failure to complete autophagy in hearts from (E) 14 and 6 mice for LC3-II and Gabarap-II, respectively; and (G) 5-6 mice except for Atg4B (11 mice) and Atg3 (16 mice). (H) Lysosomal cathepsin D (Ctsd) and its cleaved intermediate were elevated in hearts from Tfrl^^mice, indicating normal lysosomal function. (I) Lpinl mRNA was decreased in hearts from Tfrl^^mice. (J) Optineurin (Optn) mRNA was increased in hearts from Tfrl^^mice. Data were presented as means ± SEM. Sample size (n) was indicated. *p < 0.05, **p < 0.01, and ***p < 0.001 by one-way ANOVA; n.s. was not significant. See also FIG. 9.

[0017] Figure 6 shows attempts to rescue Tfrl^^mice. (A) R654A Tfrl prolonged lifespan of Tfirl mice by several days. P-value was determined by the LogRank test. Sample size (n) was indicated. (B,C) Similar to Tfrl^^mice, Tfrl^^mice constitutively expressing R654A Tfrl were iron deficient and accumulated p62 in the heart, but appeared to have normal levels of LC3-II and AtglO. (D,E) Iron overload achieved by Fe dextran administration at P3 and an Hjv- /- hemochromatosis background provided sufficient iron to fully rescue Tfrl^^mice, restoring Fe-S proteins Dpyd and Ppat as well as the iron storage protein H-ferritin and ETC complexes at 10 weeks of age. (F) There was no difference in markers of autophagy between iron-loaded Tfrl^^mice and their similarly iron-loaded littermate controls at 10 weeks of age. See also FIG. 10.

[0018] Figure 7 shows Tfrl^^mice were transiently rescued by treatment with nicotinamide riboside (NR). (A) mRNA encoding Nmrk2/ltgblbp3 was massively increased in Tfrl^^mice. (B) mRNAs encoding Slc3a2 and Slc7a5, components of the uptake system for tryptophan, an NAD precursor, were increased in Tfrl^^mice. (C) mRNAs encoding ADP-ribosyltransferases Artl, Art4, and Art5 were decreased in Tfrl^^mice. (D) Mitochondrial proteins from hearts of Tfrl^^mice showed increased lysine acetylation. (E) Administration of the NAD precursor and Nmrk2 substrate NR extended the lifespan of Tfirl ^Mhrl mice for up to 5 days. Data were presented as means ± SEM. P-values for (A) to (C) were determined by one- way ANOVA. Sample size (n) was indicated; *p < 0.05 and ***p < 0.001. P-vale for (E) was determined by LogRank test.

[0019] Figure 8 shows the floxed Tfirc allele configuration and preliminary characterization of Tfrl^^mice (related to FIG. 1). (A) Schematic diagram of floxed Tfirc allele allowing for deletion of exons 3-6. (B) Levels of Tfirc mRNA in hearts from littermate wild-type (WT) and Tfrl^mice at P10. (C) Body weights of littermate WT and Tfrl^mice at P10. Data were presented as means ± SEM. P-values were determined by ANOVA. The sample size (n) was indicated. ***p < 0.001; n.s., not significant.

[0020] Figure 9 shows immunoblot detection of proteins involved in autophagy and mitophagy in hearts from WT and Tf _r i ^1111/1111 _m i _ce (related to FIG. 5). (A-I) Multiple autophagy- and mitophagy -related proteins were examined by immunoblotting 10-day old littermate heart samples as indicated in each panel. (J) Normalized levels of p62 (Sqstml) mRNA were similar in hearts from WT and χβ-ι ^1111/1111 Httermates at 10 days of age. (K) Immunoblot showing increased Ctsd in hearts from χβ-ι ^1111/111 mice. Data were presented as means ± SEM. P-values were determined by one-way ANOVA. The sample size (n) was indicated; n.s., not significant.

[0021] Figure 10 shows ongoing iron loading was necessary to rescue Tf _r i ^1111/1111 (related to FIG. 6). (A) Amounts of Fe-S cluster proteins Dpyd and Ppat in hearts from 10-day old WT and jf _r1 ^h r ^t/h r ^t _m j _{ce un} t _rea t _ec j _or treated with iron dextran. (B) Representative ETC complex proteins in hearts from 10-day old WT and χβ-ι ^{1111 1111} mice untreated or treated with iron dextran. (C) Heart weight to body weight ratios at P10 for WT and χβ-ι ^{1111 1111} httermates treated with 1 dose of iron dextran treated on P3. (D) Representative ETC complex proteins in hearts from 6-8 week old WT and Tfirl ^MhA mice treated with 2 doses of iron dextran (at P3 and P7). (E) Heart weight to body weight ratios at 6-8 weeks from WT and xfri ^{1111 1111} mice treated with 2 doses of iron dextran (at P3 and P7). (F) Heart to body weight ratios at 10-11 weeks from Tfrl ⁰⁷ ^; Hjv ^v" and

Yf _r1 ^h r ^t/h r ^t . jjj _v ^"/" _m j _ce treated with iron dextran only at P3. Data were presented as means ± SEM. P-values were determined by one-way ANOVA. The sample size (n) was indicated. *p < 0.05; n.s., not significant.

[0022] Figure 11 shows a schematic illustrating the generation of χβ-ι ^ ¹¹¹¹

[0023] Figure 12 shows images of heart sections from wild-type (WT) and χβ-ι ^ ¹¹¹¹ mice.

[0024] Figure 13 shows end diastolic diameter (EDD) and end systolic diameter (ESD) from wild-type (WT) mice.

[0025] Figure 14 shows relative protein content for (A) LC3-II, (B) p62, and (C) AtglO in wild-type (WT; white bars) and Tfrl ^MhA (gray bars).

[0026] Figure 15 shows a schematic illustrating complexes I, II, III, and IV of the electron transport chain (ETC).

[0027] Figure 16 shows a schematic of nicotinamide riboside (NR) feeding into the pathway for synthesizing NAD.

[0028] Figure 17 shows a schematic of how deletion of Tfrl in the heart leads to iron deficiency, which in turn, leads to mitochondrial dysfunction and defective mitophagy. Defective mitophagy did not allow for the recovery of iron from dysfunctional mitochondria.

[0029] Figure 18 shows abnormal mitochondrial morphology and function in hearts from

T _{fr l} hrt/h ⁱ t _mice ^ _{Enzymatic ac} tivity of complex II of ETC from P5 τ^ ^ and WT littermates. (B) Enzymatic activity of complexes I to IV of ETC from P10 xf _r ⁱ ^ ¹¹¹ _a nd WT littermates. (C) Enzymatic activity of complex V of ETC from P10 Tfrl and WT littermates.

[0030] Figure 19 shows the levels of UPR ^MT mRNAs in hearts from WT and Tfr 1 mice untreated (A) or treated with NR (B). NR treatment appears to have blunted the UPRMT response.

[0031] Figure 20 shows p62 protein levels in hearts from WT and Tf _r i ^ ¹¹¹¹ _m i _ce that were untreated (A) or treated with NR (B).

DETAILED DESCRIPTION

[0032] The present invention relates to a method for treating heart failure in a subject in need thereof. The heart failure may be associated with iron deficiency. The method includes administering nicotinamide riboside to the subject. Nicotinamide riboside is acted upon in the subject to induce the production of NAD, which in turn, increases the lifespan of the subject. The lifespan of the subject may be increased as compared to a lifespan of a subject suffering from heart failure and not administered nicotinamide riboside.

[0033] Nicotinamide riboside may also enhance or increase the mitochondrial unfolded- protein response, and thus, the method may enhance or increase the mitochondrial unfolded- protein response in the subject as compared to a mitochondrial unfolded-protein response in a subject suffering from heart failure and not administered nicotinamide riboside. Alternatively, nicotinamide riboside may inhibit or decrease the mitochondrial unfolded- protein response, and thus, the method may inhibit or decrease the mitochondrial unfolded- protein response in the subject as compared to a mitochondrial unfolded-protein response in a subject suffering from heart failure and not administered nicotinamide riboside.

[0034] The method may also include administering iron to the subject and thus, provides a combination therapy of nicotinamide riboside and iron for the subject suffering from heart failure.

[0035] The present invention also relates to a method of identifying a subject suffering from heart failure as a candidate for treatment with nicotinamide riboside.

1. Definitions

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0037] The terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments "comprising," "consisting of and "consisting essentially of," the embodiments or elements presented herein, whether explicitly set forth or not.

[0038] "Heart failure" as used herein refers to a chronic, progressive condition in which the heart muscle is unable to pump enough blood to meet the subject's needs for blood and oxygen. Heart failure is a clinical disorder characterized by congestion and decreased functional capacity. In some embodiments, the heart failure may be characterized by or associated with iron deficiency. Heart failure may include the left side, right side, or both sides of the heart.

Accordingly, in some embodiments, heart failure may be left-sided heart failure (systolic failure or diastolic failure), right-sided heart failure, congestive heart failure, or any combination thereof. The heart failure may be classified by the New York Heart Association (NYHA) Functional Classification system. The NYHA Functional Classification system may include a functional capacity, which is a description of how the subject feels during physical activity, and an objective assessment. Functional capacity is ranked from class I to class IV, with increasing limitations of physical activity. Objective assessment is ranked from class A to class D, with increasing severity.

[0039] "Iron deficiency," "Sideropenia," and "Hypoferremia" as used herein may be used interchangeably to describe a condition in which a subject has a less than normal supply of iron. A normal Total Iron Binding Capacity (TIBC) test should range from 240 mcg/dL to 450 mcg/dL, resulting in a normal transferrin saturation range of 20 percent to 50 percent.

[0040] Iron deficiency may be accompanied with anemia, but need not be accompanied by anemia. Iron deficiency may exacerbate heart failure even when anemia is not present in the subject. In some embodiments, a subject suffering from heart failure may be considered iron deficient if ferritin is less than 100 μg/L, iron is less than 6 μπιοΙ/L, transferrin is less than 25 μπιοΙ/L, or transferrin saturation is less than 20%.

[0041] In some embodiments, the presence or absence of iron deficiency in the subject may be determined by examining red blood cells from the subject for characteristics of iron deficiency. In other embodiments, the presence or absence of iron deficiency in the subject may be determined by measuring the ratio of serum iromtotal iron-binding capacity; this ratio is a dynamic measurement of how much iron is available to the tissues of the subject. In still other embodiments, the presence or absence of iron deficiency in the subject may be determined by measuring the level of serum ferritin in a sample obtained from the subject. [0042] "Sample," "test sample," "specimen," "sample from a subject," and "patient sample" as used herein may be used interchangeably and may be a sample of blood, tissue, urine, serum, plasma, amniotic fluid, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes, or monocytes. The sample can be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.

[0043] Any cell type, tissue, or bodily fluid may be utilized to obtain a sample. Such cell types, tissues, and fluid may include sections of tissues such as biopsy and autopsy samples, frozen sections taken for histologic purposes, blood (such as whole blood), plasma, serum, sputum, stool, tears, mucus, saliva, bronchoalveolar lavage (BAL) fluid, hair, skin, red blood cells, platelets, interstitial fluid, ocular lens fluid, cerebral spinal fluid, sweat, nasal fluid, synovial fluid, menses, amniotic fluid, semen, etc. Cell types and tissues may also include lymph fluid, ascetic fluid, gynecological fluid, urine, peritoneal fluid, cerebrospinal fluid, a fluid collected by vaginal rinsing, or a fluid collected by vaginal flushing. A tissue or cell type may be provided by removing a sample of cells from an animal, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose). Archival tissues, such as those having treatment or outcome history, may also be used. Protein or nucleotide isolation and/or purification may not be necessary.

[0044] "Subject" and "patient" as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgous or rhesus monkey, chimpanzee, etc.) and a human). In some

embodiments, the subject may be a human or a non-human. The subject or patient may be undergoing other forms of treatment.

[0045] "Treat", "treating" or "treatment" are each used interchangeably herein to describe reversing, alleviating, or inhibiting the progress of a condition or disease, or one or more symptoms of such condition or disease, to which such term applies. Depending on the subject, the term also refers to preventing a condition or disease, and includes preventing the onset of a condition or disease, or preventing the symptoms associated with a condition or disease. A treatment may be either performed in an acute or chronic way. The term also refers to reducing the severity of a condition or disease, or symptoms associated with such condition or disease prior to affliction with the condition or disease. Such prevention or reduction of the severity of a condition or disease prior to affliction refers to administration of nicotinamide riboside with or without iron, or a pharmaceutical composition of the present invention to a subject that is not at the time of administration afflicted with the condition or disease. "Preventing" also refers to preventing the recurrence of a condition or disease or of one or more symptoms associated with such condition or disease. "Treatment" and "therapeutically," refer to the act of treating, as "treating" is defined above.

[0046] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

2. Method of Treatment

[0047] Provided herein is a method of treating heart failure in a subject in need thereof. The heart failure may be associated with iron deficiency. The method may include administering nicotinamide riboside (NR) to the subject. The method may further comprise administering iron to the subj ect.

[0048] The subject may have iron deficiency. The subject may have iron deficiency in the heart. The subject may have iron deficiency with anemia. The subject may have iron deficiency without anemia.

[0049] The subject may have dysfunctional mitochondria. The subject may have

dysfunctional mitochondria in the heart. The dysfunctional mitochondria may be caused by iron deficiency in the subject. In turn, the dysfunctional mitochondria may cause a reduced

NAD/NADH ratio in the subject.

[0050] The subject may have reduced mitophagy. The subject may have dysfunctional mitophagy. The subject may have dysfunctional mitophagy in the heart. The dysfunctional mitophagy may be caused by iron deficiency in the subject, but may also exacerbate the iron deficiency in the subject because iron may not be recovered from dysfunctional mitochondria.

[0051] The subject may have a decreased NAD/NADH ratio as compared to a NAD/NADH ratio of a subject not suffering from heart failure. In some embodiments, the subject may have a decreased NAD/NADH ratio in the heart as compared to a NAD/NADH ratio in the heart of the subject not suffering from heart failure.

[0052] In some embodiments, the subject may have a decreased NAD/NADH ratio as compared to a NAD/NADH ratio of a subject not suffering from heart failure associated with iron deficiency. In other embodiments, the subject may have a decreased NAD/NADH ratio in the heart as compared to a NAD/NADH ratio in the heart of the subject not suffering from heart failure associated with iron deficiency.

[0053] The method may enhance or increase the mitochondrial unfolded-protein response in the subject. The method may enhance or increase the mitochondrial unfolded-protein response in the heart of the subject. The method may enhance or increase the mitochondrial unfolded- protein response in the subject as compared to a mitochondrial unfolded-protein response in a subject suffering from heart failure and not administered nicotinamide riboside. The enhanced or increased mitochondrial unfolded-protein response may correlate with an increase in expression of one or more UPR ^MT mRNAs. The method may inhibit or decrease the mitochondrial unfolded-protein response in the heart of the subject. The method may inhibit or decrease the mitochondrial unfolded-protein response in the subject as compared to a mitochondrial unfolded- protein response in a subject suffering from heart failure and not administered nicotinamide riboside. The inhibited or decreased mitochondrial unfolded-protein response may correlate with decrease or inhibition of expression of one or more UPR ^MT mRNAs.

[0054] The method may prolong the life span of the subject as compared to a lifespan of a subject suffering from heart failure and not treated with the method described herein. The method may prolong the life span of the subject by up to about 50% as compared to the lifespan of the subject suffering from heart failure and not treated with the method described herein. The method may prolong the life span of the subject by up to about 5% to about 50%, about 10%> to about 50%, about 15% to about 50%, about 20% to about 50%, about 25% to about 50%, about 30% to about 50%, about 35% to about 50%, about 40% to about 50%, about 5% to about 45%, about 5%) to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 45%), about 15% to about 40%, or about 20% to about 35% as compared to the lifespan of the subject suffering from heart failure and not treated with the method described herein. The method may prolong the lifespan of the subject by up to about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% as compared to the lifespan of the subject suffering from heart failure and not treated with the method described herein.

[0055] The method may extend the lifespan of the subject as compared to the lifespan of the subject suffering from heart failure and not treated with the method described herein. The method may extend the lifespan of the subject by at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks, 51 weeks, 52 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, 36 months, 37 months, 38 months, 39 months, 40 months, 41 months, 42 months, 43 months, 44 months, 45 months, 46 months, 47 months, 48 months, 49 months, 50 months, 51 months, 52 months, 53 months, 54 months, 55 months, 56 months, 57 months, 58 months, 59 months, 60 months, 1 year, 2 years, 3 years, 4 years, or 5 years as compared to the lifespan of the subject suffering from heart failure and not treated with the method described herein. a. Nicotinamide Riboside

[0056] As described above, the method of treating heart failure may include administering nicotinamide riboside to the subject. Nicotinamide riboside is a form of vitamin B3 and may be phosphorylated by Nmrk proteins to induce NAD production. Induction of NAD production may, in turn, increase the NAD/NADH ratio in the subject administered nicotinamide riboside. Administration of nicotinamide riboside may also increase the lifespan of the subject and the mitochondrial unfolded-protein response in the subject as described above. Alternatively, administration of nicotinamide riboside may decrease the mitochondrial unfolded-protein response in the subject as described above.

[0057] In some embodiments, the method may administer a therapeutically effective amount of nicotinamide riboside to the subject. The method may administer about 500 mg/day to about 2000 mg/day, about 550 mg/day to about 1900 mg/day, about 600 mg/day to about 1800 mg/day, about 650 mg/day to about 1700 mg/day, about 700 mg/day to about 1600 mg/day, about 750 mg/day to about 1500 mg/day, about 800 mg/day to about 1400 mg/day, about 850 mg/day to about 1300 mg/day, about 900 mg/day to about 1200 mg/day, about 950 mg/day to about 1100 mg/day, about 500 mg/day to about 1900 mg/day, about 500 mg/day to about 1800 mg/day, about 500 mg/day to about 1700 mg/day, about 500 mg/day to about 1600 mg/day, about 500 mg/day to about 1500 mg/day, about 500 mg/day to about 1400 mg/day, about 500 mg/day to about 1300 mg/day, about 500 mg/day to about 1200 mg/day, about 500 mg/day to about 1100 mg/day, about 500 mg/day to about 1000 mg/day, about 500 mg/day to about 950 mg/day, about 500 mg/day to about 900 mg/day, about 500 mg/day to about 850 mg/day, about 500 mg/day to about 800 mg/day, about 500 mg/day to about 750 mg/day, about 500 mg/day to about 700 mg/day, about 500 mg/day to about 650 mg/day, about 500 mg/day to about 600 mg/day, about 550 mg/day to about 2000 mg/day, about 600 mg/day to about 2000 mg/day, about 650 mg/day to about 2000 mg/day, about 700 mg/day to about 2000 mg/day, about 750 mg/day to about 2000 mg/day, about 800 mg/day to about 2000 mg/day, about 850 mg/day to about 2000 mg/day, about 900 mg/day to about 2000 mg/day, about 950 mg/day to about 2000 mg/day, about 1000 mg/day to about 2000 mg/day, about 1100 mg/day to about 2000 mg/day, about 1200 mg/day to about 2000 mg/day, about 1300 mg/day to about 2000 mg/day, about 1400 mg/day to about 2000 mg/day, about 1500 mg/day to about 2000 mg/day, about 1600 mg/day to about 2000 mg/day, about 1700 mg/day to about 2000 mg/day, about 1800 mg/day to about 2000, or about 1900 mg/day to about 2000 mg/day nicotinamide riboside to the subject.

[0058] In other embodiments, the method may administer about 500 mg/day, 550 mg/day, 600 mg/day, 650 mg/day, 700 mg/day, 750 mg/day, 800 mg/day, 850 mg/day, 900 mg/day, 950 mg/day, 1000 mg/day, 1100 mg/day, 1200 mg/day, 1300 mg/day, 1400 mg/day, 1500 mg/day, 1600 mg/day, 1700 mg/day, 1800 mg/day, 1900 mg/day, or 2000 mg/day nicotinamide riboside to the subj ect. b. Iron

[0059] As described above, the treatment method may further administer iron to the subject. The iron may be administered to the subject in a therapeutically effective amount. The iron may be administered to the subject in an amount such that circulating transferrin (Tf) is further saturated with iron. Transferrin is a protein found in the blood and binds and transports iron throughout the bodies of mammals.

[0060] The iron administered to the subject may in the form of ferrous sulfate, ferrous fumarate, ferrous gluconate, ferric carboxymaltose, ferric saccharate, ferric gluconate, iron dextran, iron sucrose, or any combination thereof.

[0061] The iron may be administered orally to the subject, for example, in tablet form. In some embodiments, the method may administer to the subject an oral daily dose from about 150 mg/day to about 200 mg/day of elemental iron. In other embodiments, the method may administer an oral daily dose from about 100 mg/ day to about 300 mg/day or about 125 mg/day to about 250 mg/day of elemental iron. In some embodiments, the method may administer to the subject an oral daily dose of about 100 mg/day, 105 mg/ day, 1 10 mg/day, 1 15 mg/day, 120 mg/day, 125 mg/day, 130 mg/day, 135 mg/day, 140 mg/day, 145 mg/day, 150 mg/day, 155 mg/day, 160 mg/day, 165 mg/day, 170 mg/day, 175 mg/day, 180 mg/day, 185 mg/day, 190 mg/day, 195 mg/day, 200 mg/day, 205 mg/day, 210 mg/day, 215 mg/day, 220 mg/day, 225 mg/day, 230 mg/day, 235 mg/day, 240 mg/day, 245 mg/day, 250 mg/day, 255 mg/day, 260 mg/day, 265 mg/day, 270 mg/day, 275, mg/day, 280 mg/day, 285 mg/day, 290 mg/day, 295 mg/day, or 300 mg/day of elemental iron.

[0062] The iron may be administered intravenously to the subject. In some embodiments, the method may administer to the subject up to about 1000 mg of elemental iron per week. In other embodiments, the method may administer to the subject up to about 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, or 950 mg of elemental iron per week. c. Combinatorial Therapy

[0063] As described above, the treatment method administers nicotinamide riboside to the subject and may further include administering iron to the subject. The nicotinamide riboside and iron may be administered simultaneously. In other embodiments, the nicotinamide riboside and iron may be administered sequentially, in which nicotinamide riboside administration may precede iron administration or iron administration may precede nicotinamide riboside

administration. d. Pharmaceutical Composition

[0064] The nicotinamide riboside and/or iron may be incorporated into a pharmaceutical composition suitable for administration to a subject (such as a patient, which may be a human or non-human).

[0065] The pharmaceutical composition may include a "therapeutically effective amount" or a "prophylactically effective amount" of nicotinamide riboside and/or iron. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of nicotinamide riboside and/or iron may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of nicotinamide riboside and/or iron are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount.

[0066] The pharmaceutical composition may include pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier," as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, corn starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt;

gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

3. Diagnostic Method

[0067] Also provided herein is a method for identifying a subject suffering from heart failure as a candidate for treatment with nicotinamide riboside. In some embodiments, the method may also identify a subject suffering from heart failure associated with iron deficiency as a candidate for treatment with nicotinamide riboside. In still other embodiments, the method may further identify a subject suffering from heart failure associated with iron deficiency as a candidate for treatment with nicotinamide riboside, in which anemia is present with the iron deficiency.

[0068] The method may include obtaining a sample from the subject and measuring in the sample a level of a ratio of NAD/NADH, a level of iron, a level of a secreted or serum protein marker, or any combination thereof.

[0069] The secreted or serum protein marker may be secreted from a heart cell such as a cardiomyocyte. The secreted or serum protein marker may be secreted from a cell having the mitochondrial unfolded-protein response. The secreted or serum protein marker may be FGF21 protein, or Angiopoietin-like 4 (AngPtL4) protein, or the combination thereof. Accordingly, the method may measure a level of FGF21, AngPtL4, or the combination thereof.

[0070] Measuring the level of iron may include measuring serum ferritin, transferrin, transferrin saturation, serum iron, total iron-binding capacity, or any combination thereof.

[0071] The method may also include comparing the measured level to a control level. In some embodiments, the control level may be a predetermined or reference value, which is employed as a benchmark against which to assess the measured result. The predetermined or reference value may be a level in a sample from a subject not suffering from heart failure.

[0072] The method may further include identifying the subject as a candidate for treatment with nicotinamide riboside if measured level of the ratio of NAD/NADH is lower than the control level of the ratio of NAD/NADH, the measured level of iron is lower than the control level of iron, the measured level of FGF21 protein is higher than the control level of FGF21 protein, the measured level of AngPtL4 protein is higher than the control level of AngPtL4 protein, or any combination thereof. The nicotinamide riboside may be administered, alone or in combination with iron as described above, to the subject identified as a candidate for treatment with nicotinamide riboside.

4. Diagnostic Kit

[0073] Also provided herein a kit which may be used with the diagnostic method described above. The kit may include a positive control and/or a negative control. The kit may include material(s), which may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and/or other material useful in sample processing, washing, or conducting any other step of the diagnostic method described herein.

[0074] The kit according to the present disclosure may also include instructions for carrying out the diagnostic method. Instructions included in the kit of the present disclosure may be affixed to packaging material or may be included as a package insert. While instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term "instructions" can include the address of an internet site which provides instructions.

[0075] The present invention has multiple aspects, illustrated by the following non-limiting examples. 5. Examples

Example 1

Materials and Methods for Examples 2- 7

[0076] Animal Experiments. 129/SvEv mice bearing a floxed Tfrc allele were crossed with transgenic C57BL/6 mice expressing Cre recombinase under the control of the a-MyHC promoter. Backcrossing was with 129/SvEv mice for more than 10 generations and after the initial phenotypic characterization, all studies used mice with a homogeneous 129/SvEv background. Animals were genotyped by polymerase chain reaction (PCR) using genomic DNA from toe clips. Primers for Tfrc alleles, Cre, Hjv alleles, and Rosa26 are described in Table 1.

[0077] Table 1 : Primers for genotyping.

[0078] For iron rescue experiments, mice were injected intraperitoneal (IP) with 5 mg of iron dextran (Uniferon® 100 (25 μΐ)) at P3, or at both P3 and P7. For nicotinamide riboside (NR) rescue experiments, mice were injected IP with NR dissolved in PBS at 750 mg/kg daily from P5. Volume was based on the body weight, with less than 100 μΐ at postnatal day 10. Control animals were injected with PBS only as described in Yang et al. (2007) J. Med. Chem. 50:6458- 6461, the entire contents of which are incorporated herein by reference.

Histopathological Analysis. 4μιη paraffin sections were stained with hematoxylin and eosin and examined by light microscopy. Cardiomyocyte dimensions were measured by digital

morphometry of paraffin-embedded myocardial cross-sections stained with Alexa Fluor® 594 WGA (Life Technologies, Invitrogen) using ImageJ image processing. Apoptotic cells were identified and quantified using the In Situ Cell Death Detection Kit (Roche) on paraffin sections according to the manufacturer's instructions. Quantitation was done by ImageJ using three consecutive sections for each mouse.

[0079] Echocardiography. Echocardiography was performed without anesthesia on 7 or 8 age-, sex- and body weight-matched mice per genotype by an investigator blinded to genotypes. Left ventricular dimension and fractional shortening were calculated based on data generated by echocardiography as described in Esposito et al. (2000) Am. J. Physiol. Heart Circ. Physiol. 279 :H3101-3112, the entire contents of which are incorporated herein by reference.

[0080] Electron Microscopy . Hearts were removed and rinsed quickly in cold Krebs- Henseleit buffer, and then immersed in 5% glutaraldehyde buffer. Samples were prepared for transmission electron microscopy and imaged by Duke University Medical Center Research Electron Microscopy Services.

[0081] Tissue Iron Analysis. Heart non-heme iron was measured as described in Levy et al. (1999) Nat. Genetics 21 :396-399 and Torrance and Bothwell (1980) Methods in Hematology,

[0082] J.D. Cook, ed. (New York, Churchill Livingstone Press) pp. 104-109, the entire contents of both of which are incorporated herein by reference.

[0083] Enzymatic Analysis of Respiratory Chain Complexes I-IV. Tissues were homogenized in 250 mM sucrose, 40 mM potassium chloride, 1 mM EGTA, 1 mg/ml fatty acid free BSA, 20 mM Tris-HCl (pH 7.2) and homogenates were centrifuged at 600 x g for 10 min at 4°C. Steady state activities of enzyme complexes in the supernatant were determined as described in Janssen et al. (2007) Clin. Chem. 53 :729-734 and Spinazzi et al. (2011) Mitochondrion 11 :893-904, the entire contents of both of which are incorporated herein by reference.

[0084] Preparation of Mitochondrial Ly sates and Acetyl-lysine Analysis. Mouse hearts were homogenized (40-50 strokes) in 15 volumes of ice-cold homogenization buffer [320 mM sucrose, 50 mM KH2P04 (pH 7.4), 10 mM Tris-HCl (pH 7.4), 1 mM EDTA] in the presence of protease inhibitors, phosphatase inhibitors (Roche), and deacetylase inhibitors (2.5 μΜ

Trichostatin A, 5 mM nicotinamide, 5 mM sodium butyrate) using glass homogenizers. Crude mitochondria were isolated by differential centrifugation. Homogenates were centrifuged at 1600 rpm for 10 min at 4°C. The supernatant was centrifuged again at 1600 rpm for 10 min at 4°C. The supernatant was subsequently centrifuged at 10,000 x g for 10 min at 4°C. The pellet was collected, rinsed with 1 ml homogenization buffer and centrifuged at 10,000 x g again for 10 min at 4°C. The pellet was resuspended in 100 μΐ 20 mM HEPES (pH 7.4), 150 mM NaCl, and 1% Triton-X-100 with protease, phosphatase and deacetylase inhibitors. Western blot analysis was performed with 70μg protein per lane. Antibodies recognizing acetyl-lysine were purchased from Cell Signaling Technology.

[0085] Statistical Analysis. One-way ANOVA was performed for comparisons between two means, including when the comparison was between the means of WT and Tfrl ^{1111 1111} . Survival analysis was performed using Logrank in Graphpad software. P < 0.05 was considered statistically significant.

[0086] An independent statistical analysis for survival analysis was also performed as described below.

[0087] Survival time was modeled as follows:

[0088] Tij = μ + β] + eij

[0089] Where Tij was the survival time (in days) for the i-th mouse in the j-th group. The model explained this in terms of a baseline mean m, an effect bj for the j-th group (relative to baseline) and inter-mouse variation eij . The model was fit using ordinary least squares (1-way ANOVA).

[0090] For the TfrlR654A rescue experiment, the baseline was xf _r i ^ ¹¹¹ * There was a significant improvement in mean survival in the Jftl ^ TfrlR654A group (2.7 days, p-value < 0.001, Table 2). In WT, the improvement was over 49 days (all observations were censored in this group).

[0091] Table 2: Analysis of survival for experiment 2.

[0092] For the R rescue experiment, the baseline was Tfrl with PBS. There was no significant improvement in mean survival when using just χβ-ι ^ ¹¹¹¹ (0.625 days, p-value = 0.26, Table 3). There was, however, a significant improvement in the χβ-ι ^ ¹¹¹¹ with NR group (2.29 days, p-value < 0.001). For statistical calculations, lifespan was over 19.6 days for wild-type mice.

[0093] Table 3 : Analysis of survival for experiment 1.

[0094] Western Blot Analysis. Mouse hearts were homogenized in 15 volumes of ice-cold RIPA buffer in the presence of protease inhibitors and phosphatase inhibitors (Roche) using glass homogenizers. Protein lysates were collected as the supernatant after centrifugation at 14,000 rpm for 15 min at 4 °C. Protein concentrations were determined using the DC protein assay (Bio-Rad). Antibodies were used recognizing Dpyd, Cathepsin D, Tom20 (Santa Cruz Biotechnology), LC3, Gabarap, p62, Beclin-1, Ulkl, Atg3, Atg4B, Atg7, Atg5, Atgl2, Atgl6Ll, Bnip3, Nix, H-Ferritin (Cell Signaling Technology), AtglO (ThermoFisher Scientific), Fundcl (Aviva Systems Biology), Rcanl (Abgent), Rpll9 (Sigma- Aldrich), Tfrl (Invitrogen), Ppat and Atg9A (Novus Biologicals). Total OXPHOS Rodent WB Antibody Cocktail was purchased from Abeam.

[0095] RNA Extraction, Microarray Gene Expression Analysis, and PCR. Total heart

RNA was isolated from flash-frozen tissue using the RNeasy Fibrous Tissue Mini Kit (Qiagen) and reverse transcribed as described in Schmidt et al. (2008) Cell Metab. 7:205-214, the entire contents of which are incorporated herein by reference. For the microarray analysis, the

Affymetrix GeneChip® Mouse Genome 430 2.0 Array was used. A minimum of 200 ng RNA was used for each sample. RNA samples were processed by the Duke Microarray Shared Resource Services. Transcript abundance was quantified by 2-step real-time PCR, using amplification conditions as described in Schmidt et al. (2008) Cell Metab. 7:205-214, the entire contents of which are incorporated herein by reference. Primers are summarized in Table 2 and Rpl l9 was used as an internal control.

[0096] Table 4: Primers for real-time.

[0097] Spectrophotometry Analysis of Single Respiratory Chain Complexes I-IV. All reactions were performed using a computer-tunable spectrophotometer (Spectramax Plus Microplate Reader, Molecular Devices, Sunnyvale, CA) at 20-s intervals for 5 minutes at room temperature. The reaction conditions for assaying activity of each complex were described in Janssen et al. (2007) Clin. Chem. 53 :729-734 and Spinazzi et al. (2011) Mitochondrion 11 :893- 904, the entire contents of both of which are incorporated herein by reference. The total volume of each reaction was 200 μΐ. Each sample had three replicates for total activity and inhibitor- insensitive activity. For complexes I and II, 2,6-dichloroindophenol (DCIP) reduction by electrons accepted from decylubiquinol was measured. The reduction of DCIP was followed by spectrophotometry at 600 nm. 20 μg heart homogenates were used in each reaction for both complex I and complex II activity assays. Complex III activity was measured by following the reduction of cytochrome c at 550 nm. Complex IV activity was measured by following the oxidation of reduced cytochrome c at 550 nm. 3.6 μg and 1 μg of heart homogenate were used in each reaction for complex III and complex IV activity assays, respectively. The addition of standard respiratory chain inhibitors for each complex was used to ascertain the specificity of the enzymatic assays. The same amount of protein was used for each sample. Example 2

Tfrl Deficiency Causes Cardiomyopathy

[0098] Murine Tfrl was inactivated in cardiomyocytes by expressing aMyh6-Cre transgene to recombine loxP sites flanking Tfrc exons 3 to 6 (FIGS. 8A and 11). It was confirmed that mutant animals (Tfrl ^11111111 ) expressed little Tfrl mRNA in heart (FIG. 8B). Tfrl™ mice were born in Mendelian ratios and maintained body weights similar to wild type (WT) littermates (Tfirl^ and Tfrl^ mice, FIGS. 1 A and 8C), but died after several hours of distress by postnatal day 11 (PI 1) with cardiac hypertrophy (FIGS. IB and 12) and elevated heart to body weight ratios (FIG. 1C), which had not been observed at P5 (not shown).

[0099] Echocardiography of Tfrl ^Mhrl mice showed left ventricular dilatation in both systole and diastole, and decreased fractional shortening (FIGS. ID and IE), indicating compromised performance of the heart as a pump. FIG. 13 shows the end diastolic diameter (EDD) and end systolic diameter (ESD) from wild-type (WT) mice. Wheat germ agglutinin staining showed enlarged χβ-ι ^ ⁷¹¹¹¹ cardiomyocytes, consistent with cardiomyocyte hypertrophy (FIG. IF). There was increased mRNA encoding markers for cardiac hypertrophy Nppa (atrial natriuretic peptide) and Nppb (brain natriuretic peptide), My 7 (myosin heavy chain β), and Actal (FIG. 1G). Thus, deletion of Tfrl in cardiomyocytes led to dilated cardiomyopathy and early death.

Example 3

Cardiac Iron Deficiency

[00100] The canonical function of Tfrl is to supply iron to meet cellular needs. Non-heme cardiac iron was measured to determine whether loss of Tfrl resulted in iron deficiency. At P5, cardiac iron concentration was decreased in χβ-ι ^ ¹¹¹ mice. Between P5 and P10, non-heme iron nearly doubled in WT hearts, but increased only slightly in Tfrl ^MhA hearts (FIGS. 2 A and 2B), indicating that loss of Tfrl impaired iron uptake. The total iron concentration at P10 showed less deficit in χβ-ι ^ ¹¹¹ _m i _ce (FIG. 2C), indicating that heme iron was relatively spared. Fe-S clusters are synthesized from non-heme iron, and the amounts of enzymes Dpyd and Ppat are decreased when Fe-S clusters were not available. Both proteins were deficient in Tfrl™ hearts (FIG. 2D), consistent with compromised Fe-S cluster biogenesis due to iron deficiency or mitochondrial dysfunction. It was concluded that Tfrl was important for iron uptake by cardiomyocytes. Example 4

Metabolic Changes Associated with

Cardiomyopathy

[00101] Mitochondria from JM™^ hearts were slightly abnormal at P5, and severely disrupted and enlarged at P10 (FIGS. 3 A and 14). Fe-S clusters and heme are required by most complexes of the electron transport chain (ETC). FIG. 15 shows a schematic illustrating the requirement of Fe-S clusters and heme by complexes I, II, III, and IV of the ETC. As such, ETC complexes were examined by immunoblotting for proteins that are labile when the complexes are not assembled properly.

[00102] At P5, complex II was decreased in χβ-ι ^ ¹¹¹¹ hearts and complex IV was increased, but the other ETC complexes did not differ from controls (Fig 3B).

[00103] Iron-containing complexes I through IV were diminished in hearts from xf _r i ^ ¹¹¹¹ _m i _ce at P10, but complex V, which does not contain iron, was unchanged (FIG. 3C). Accordingly, activities of complexes I to IV were markedly decreased in xf _r i ^ ¹¹¹ hearts (FIG. 3C). Activity of complex II was decreased at P5 (FIG. 18 A), and activities of complexes I to IV were all markedly decreased at P10 in Tfrlhrt/hrt hearts (FIG. 18B) at P10. However, complex V, which does not contain iron, appeared unchanged at both ages, and its activity was not decreased in Tfrlhrt/hrt hearts at P10 (Fig 18C).

[00104] Expression of mitochondrial RNA polymerase (Polrmt) and mitochondria-encoded mRNA for Nd4, Cytb and Cox3 was similar to WT at P5 (not shown), but by P10 all were decreased in Tfrl hearts (FIG. 3D), indicating fewer mitochondria or mitochondria incapable of normal gene expression.

[00105] mRNA profiling was used to examine gene expression changes in χβ-ι ^ ¹¹¹¹ hearts and patterns were looked for using Gene Set Enrichment Analysis. Genes downregulated in the mutants were significantly associated with PPAR (particularly PPARa) and PGCl-a signaling, myogenesis, insulin signaling and cardiomyopathy. Upregulated genes were significant for hypoxia-inducible targets, Myc targets and glycolytic enzymes.

[00106] Decreased expression of mRNA encoding both PGCl-a {Ppargcla) and PGCl-β (Ppargclb) in Tfrl ^{hrt hlt} hearts (FIG. 3E) was confirmed. PGCl-a controls transcription of a suite of nuclear genes to induce mitochondrial biogenesis. Mice deficient in PGCl-a in the heart developed cardiomyopathy, similar to the mutant mice Tfrl . These results indicated an impaired ability to induce mitochondrial biosynthesis in χβ-ι ^ ¹¹¹¹ mice.

[00107] Cardiomyopathy is associated with a switch to fetal-like metabolism, with glucose, rather than fatty acids, as the preferred energy source. The switch has been attributed to decreased activity of PPARa {Ppara), which forms a heterodimer with Rxr. Expression of Ppara and Rxrg was decreased in Tfrl-null hearts, as was fatty acid transport protein

{Fatpl/Slc27al, FIG. 3F). Ppara expression is induced by the histone demethylase Kdm3a, which requires iron. Ppara and other genes induced by Kdm3a, Ucp2, and Acadm, had decreased mRNA levels (not shown) in xf _r i ^ ¹¹¹¹ mice. These results indicated that that iron deficiency caused decreased Ppara expression, contributing to the metabolic switch in Tfrl ^Mhrl mice.

[00108] Increased mRNA expression of hypoxia-inducible genes (FIG. 4A) and glycolytic enzymes (FIG. 4B) was observed at P10. Of the glycolytic enzymes, only Pfkl was slightly increased at P5. Iron is a cofactor for hydroxylases that cause HIFa transcriptional regulators to be degraded and inactivated, indicating that iron deficiency may be explained by these results. In addition, Myc, which also induces expression of glycolytic enzymes, was upregulated (FIG. 4C). However, glycolysis was unlikely to meet energy needs of cardiomyocytes, which depend on mitochondrial respiration. Apoptosis was increased inTfrl ^{111 111} hearts (FIG. 4D), consistent with severe mitochondrial dysfunction. Together, the results indicated that iron deficiency led to mitochondrial insufficiency, metabolic changes and increased apoptosis, contributing to cardiomyocyte hypertrophy and cardiac dysfunction in χβ-ι ^ ¹¹¹ mice.

Example 5

Interruption of Mitophagy

[00109] Accumulation of damaged mitochondria should activate mitophagy to clear dysfunctional organelles and recover iron for re-use. Glycolytic enzyme Hk2 promotes autophagy during energy deprivation and was upregulated in Tfrl ¹ ^ ¹¹¹¹ hearts (FIG. 4B). Both isoforms of Rcanl, which also induces mitophagy and protects against apoptosis due to hypoxia, were upregulated in Tfrl ^ΜΜ hearts (FIGS. 5 A and 9A).

[00110] Cardiomyocytes from young mice cannot be cultured, requiring mitophagy to be characterized using tissue samples. Expression of putative mitophagy receptors, Nix (Bnip31) and Fundcl, was decreased, rather than increased, in Tirl ¹ " ¹¹¹¹ hearts. Ulkl, a kinase that phosphorylates Fundcl for clearance of damaged mitochondria, was also decreased (FIGS. 5B, 9B, and 9C). Bnip3, a homolog of Nix that can trigger opening of mitochondrial permeability transition pore and loss of mitochondrial membrane potential, was markedly increased in mutant hearts (FIGS. 5C and 9D). Bnip3 transcription is induced by hypoxia-inducible factors, and thus, its increase was consistent with the upregulation of other hypoxia-inducible genes as described above.

[00111] Cisd2 (Naf-1), an Fe-S cluster protein associated with the mitochondrial membrane, was depleted in Tfrl^^mice (FIGS. 5D and 9E). Cisd2 is important for mitochondrial maintenance and oxidative phosphorylation and control of autophagy. Deficiency of Cisd2 should promote autophagy by liberating Beclin-1 from Bcl2. Beclin-1 levels were similar in mutant and WT hearts (FIGS. 5D and 9F), but its activity could not be assessed.

[00112] Mapllc3 (LC3) and Gabarap are also involved in the cargo recognition step of autophagy (FIGS. 5E, 5F, 9G, and 9H). LC3-II, a phosphatidylethanolamine (PE)-conjugated form of LC3, increases during active autophagy, but was decreased in heart samples from Tfrl^^mice. Gabarap-II was also decreased.

[00113] Other proteins involved in early steps of autophagy were evaluated to explore why mitophagy was ineffective (FIGS. 5G, 9G, and 91). Atgl6L, which was increased in xf _r i ^1111/1111 mice, is involved early in the formation of the phagophore, forming a complex with Atgl2 and Atg5. AtglO, which was decreased in mutant hearts, is an E2-like enzyme involved in Atgl2- Atg5 conjugation and LC3 conjugation to PE. Atgl2-Atg5 was increased in mutant hearts, though LC3-II was decreased. The Atgl2-Atg5 complex forms before conjugation of LC3. Knockdown of LC3 or Gabarap leads to maintenance of the Atgl2-Atg5 complex, which was consistent with observations described herein. Atg4b cleaves the carboxyl termini of pro-forms of LC3 and Gabarap to expose their lipidation sites, but it also de-lipidates both proteins.

Overexpression of Atg4b thus inhibits membrane localization and PE conjugation of LC3. Atg4b was increased in xf _r i ^1111/1111 hearts, contributing to decreased levels of LC3-II and Gabarap-II. Atg7, an El -like enzyme for ubiquitin-like conjugation systems involved in the development of autophagosomes, and Atg3, an E2-like enzyme for the LC3/Gabarap conjugation system, were both increased in Tfirl mice. Overall, these results indicated that the mutant heart cells were attempting to initiate mitophagy appropriately, but key proteins involved in cargo recognition appeared to be deficient. [00114] Sqstml (p62) is a molecule that links the phagophore to cargo. During normal autophagic flux, p62 is degraded by lysosomal enzymes. However, it was increased in xf _r i ^ ¹¹¹¹ hearts (FIGS. 5G and 9G), even though p62 mRNA was not increased (FIG. 9J), indicating that p62 accumulated because it was not degraded. Cathepsin D (Ctsd), an indicator for lysosomal function, was also examined. Both intermediate and mature forms of Ctsd were increased in hearts from Tfrl mice (FIGS. 5H and 9K), showing that lysosomes were functioning. These results indicated that a mitophagy step prior to lysosomal fusion was impaired.

[00115] Lipinl (Lpinl) enhances transcription regulated by Ppar-a and PGC-Ια and controls autophagic clearance in skeletal muscle. Lipinl deficiency in skeletal muscle leads to

accumulation of p62, similar to what was observed and described above. Lpinl mRNA was decreased in hearts from χβ-ι ^ ¹¹¹¹ _m i _ce (FIG. 51), contributing both to the metabolic switch and to the interruption of mitophagy.

[00116] Ndrgl is induced by iron depletion and was upregulated in χβ-ι ^ ¹¹¹¹ hearts (FIG. 4A). It co-localizes with Tfrl in late and recycling endosomes. Overexpression of Ndrgl accelerates Tfrl recycling and suppresses LC3-II accumulation and autophagosome formation. Although it cannot affect Tfrl in the xf _r ⁱ ^ ¹¹¹ mutants, it may contribute to the block in mitophagy.

[00117] Atg9 traffics with Tfrl and may deliver membrane components to developing autophagosomes. Atg9 was increased in Tfrl ^{hrt hrt} hearts (FIGS. 5G and 91). Optn, also important in autophagosome maturation, was markedly induced in Tf _r \ ^Mhrt hearts (FIG. 5 J). Optineurin interacts with Tfrl to modulate subcellular localization and Tf uptake. Accordingly, both Atg9 and Optn may have roles in the Tf cycle, and lack of Tfrl resulted in compensatory upregulation, perturbing normal mitophagy.

Example 6

Rescue of Tfrl ^hrt/hrt Mice

[00118] While it was observed that Tfrl deletion resulted in cardiac iron deficiency and mitochondrial dysfunction, it was also possible that the mutant phenotype resulted from abrogation of an alternative role of Tfrl, or from both loss of such a role and iron deficiency. As such, two approaches were used to address this question - it was attempted to rescue the χβ-ι ^ ¹¹¹¹ phenotype by co-expressing a Tfrl mutant protein that cannot bind Tf and, in parallel, by inducing iron overload in Tfrl mice.

[00119] Mice expressing a mutant xf _r i ^R654A protein from the ubiquitously transcribed Rosa26 locus was developed and it was shown that xf _r i ^R654A _wa s unable to bind Tf for iron uptake.

Breeding was used to obtain χΜ^χβ-ι * ⁶⁵ ^ mice, which had cardiomyocytes lacking WT Tfrl but expressing mutant Tfrl ^R654A , and compared them to xf _r i ^{fl/fl (+)} ;Xf _r i ^R654A control mice. Because R654A Tfrl cannot assimilate Tf-bound iron, hearts from χβ-ι ^χβ-ι ¹ * ⁶⁵ ^ mice should be iron deficient, but should express an otherwise intact form of Tfrl that differed from WT Tfrl by only one amino acid in the extracellular domain. If the phenotype of Tfrl ¹ ^ ¹¹¹¹ mice was due wholly or in part to loss of a function of Tfrl unrelated to iron uptake, expression of Tfrl ^R654A might fully or partially rescue the mutant phenotype.

[00120] χβ-ι»χβ-ι ^Κ654Α mice died at P13 to P14, later than mice (FIG. 6A).

Immunoblots confirmed expression of mutant Tfrl (FIG. 6B). H-ferritin and Ppat were similarly decreased in JM^^ mice at P10 and χ ^χβ-ι * ⁶⁵ ^ _m i _ce at P12, indicating that

_were i _ron deficient as expected (FIG. 6B). However, amounts of LC3-II and AtglO in hearts from χβ-ι»χί _Γ ι ^{Κ65 Α} _m i _ce were similar to χΜ ^{Μ (+)} _; χβ-ι ^Κ654Α hearts, indicating that some aspects of mitophagy were ameliorated (FIGS. 6C, 14 A, and 14C).

Nonetheless, p62 still accumulated, indicating a persistent defect in mitophagy (FIGS. 6C and 14B). These results indicated that iron deficiency was the major cause of the xf _r i ^1111/1111 phenotype. However, a contribution of Tfrl, independent of Tf binding, may explain why death of

_{Tfr l} hrt/hrt. _{Tfr l} R654A _{mi ce l ater th∑m} ^h t ^

[00121] To test whether cardiac iron repletion could rescue xf _r i ^1111/1111 mice, a large dose of iron dextran was administered at P3, to supersaturate circulating Tf and induce non-Tf-bound iron uptake. This prolonged survival, but xf _r i ^1111/1111 mice still died at 4 to 5 weeks with severe cardiomegaly. It was confirmed that the hearts had assimilated iron by immunoblotting for Fe-S cluster proteins. In contrast to untreated xf _r i ^1111/1111 mice, Dpyd and Ppat levels were similar at P10 in xf _r i ^1111/1111 _an d WT mice treated with iron dextran (FIG. 10A). Proteins representing ETC complexes were also similar at P10 (FIG. 10B). At that time, xf _r i ^1111/1111 mice and WT littermates had similar heart to body weight ratios (FIG. IOC). A second dose of iron dextran was next administered at P7. The onset of the cardiomyopathy phenotype was further delayed and Yfrihrt/hrt mice survived up to 13 weeks. However, ETC complexes were already decreased in hearts from Tfrl mice at 6-8 weeks of age (FIG. 10D) and the hearts were already enlarged (FIG. 10E). Together, these results indicated that iron-treated Tfirl ¹¹¹ ^ ¹¹¹ mice assimilated and used the supplemental iron to survive beyond their usual lifespan. However, they eventually showed abnormalities in mitochondrial ETC complexes and autophagy-related proteins similar to, but more pronounced than, untreated Tf _r i ^ ¹¹¹¹ _m i _ce at P10 (not shown).

[00122] It was hypothesized that the heart might require continuous iron uptake, and that iron administered early might no longer be available. To sustain elevated plasma iron concentrations, the hemojuvelin knockout (Hjv ^"/_ ) mice were used, which persistently have increased non-Tf- bound iron. Tfrl^^Hjv ^{" "} mice were generated, in which Tfrl was deleted in the heart and Hjv was deleted globally. These mice also died at PI 1, similar to Tfrlhrt ^/hrt mice. However, the Hjv ^" background did not allow for substantial iron accumulation during the neonatal period.

Therefore, Tfrl^Hjv ^{" "} and control mice were treated with iron dextran at P3 to support the animals until the Hjv mutation caused elevated iron levels. With this strategy, the Tfrl^Hjv ^{" "} mice had lifespans (not shown) and heart to body weight ratios (FIG. 10F) similar to Tfirl^ ⁽⁺⁾ 'Hjv ^_/" controls.

[00123] It was confirmed that this experimental protocol had restored cardiomyocyte iron by immunoblotting for Dypd and ferritin at 10-11 weeks of age (FIG. 6D). Amounts of markers for ETC complexes I to IV were also indistinguishable between Tfrl^^Hjv ^{" "} mice and controls (FIG. 6E). Autophagy-related proteins LC3-II, p62, and Beclin-1 showed no significant differences (FIG. 6F). Collectively, these results indicated that the χβ-ι ^ ¹¹¹¹ mutant phenotype was primarily attributable to a defect in iron assimilation, and iron deficiency played a critical role in development of cardiac hypertrophy, mitochondrial dysfunction and interruption of mitophagy. Importantly, it appeared that the heart was highly sensitive to iron deprivation due to inactivation of Tfrl .

Example 7

Treatment with Nicotinamide Riboside (NR)

[00124] Mitochondrial dysfunction can cause a decreased NAD/NADH ratio and inactivation of sirtuin deacetylases. A decreased NAD/NADH ratio may block signals for mitochondrial biogenesis while also causing defective mitophagy. Deacetylase activity is important for deacetylation of autophagy-related proteins; in the absence of Sirtl, LC3-II is decreased and p62 accumulates. This was similar to what was observed in χβ-ι ^1111/1111 _m i _{ce as} described above. It was hypothesized that augmentation of NAD levels might modify the mutant phenotype.

[00125] Induction of mRNA encoding nicotinamide riboside kinase 2 (Nmrk2/Itgb lbp3 ^' , FIG. 7 A) and the transport system for the NAD precursor tryptophan, Slc3a2/Slc7a5 (FIG. 7B), along with decreased expression of ADP-ribosyltransferases (FIG. 7C) was observed, indicating that mutant cardiomyocytes used multiple approaches to try to increase cellular NAD levels.

Mitochondria from xf _r i ^1111/1111 hearts showed increased lysine acetylation (FIG. 7D), consistent with decreased mitochondrial sirtuin deacetylase activity due to decreased mitochondrial NAD.

[00126] NR can be phosphorylated by Nmrk proteins to induce NAD production, activating sirtuins and mitochondrial biogenesis. How NR feeds into the synthetic pathway for NAD production is shown in FIG. 16. Noting that Nmrk2 (Itgblbp3) was markedly upregulated in Yfri ^h r ^t/h r ^t mice (FIG. 7A), it was hypothesized that this form of vitamin B3 may benefit the mutant animals, even though it should not impact iron deficiency. NR or vehicle was

administered to young animals and up to 50% prolongation of lifespans of Tfrl ^{1111 1111} mice was observed (FIG. 7E), indicating that NR contributed to NAD production and ameliorated the phenotype.

[00127] It was hypothesized that NR might improve the NAD/NADH ratio by increasing NAD. NAD/NADH ratios were measured with and without NR treatment but measurements from tissue were inconclusive. Alternatively, it was hypothesized that NR may enhance the mitochondrial unfolded protein response (UPR ^MT ). Although not apparent at P5 (not shown), increased mRNA for multiple genes associated with the UPR ^MT in P10 hearts was observed, including Atf4, Lonpl, Ddit3 and Fgf21 (FIGS. 19A and 19B). NR treatment was associated with decreased expression of UPRMT mRNAs (FIGS. 19A and 19B). Furthermore, NR alleviated the accumulation of p62 in the Tfrl hearts (FIGS. 20A and 20B).

[00128] In summary, Examples 1-7 demonstrated that mice lacking Tfrl in the heart died in the second week of life, with cardiomegaly, poor cardiac function, failure of mitochondrial respiration, and interrupted mitophagy. In this interrupted mitophagy, mitophagy was induced, but failed due to a block at the cargo recognition step. This failure of mitophagy led to iron not being recovered from dysfunctional mitochondria for re-use by cardiomyocytes. Accordingly, the interrupted mitophagy was caused by iron deficiency, but also contributed to ongoing iron deficiency (FIG. 17).

[00129] The phenotype of the mice lacking Tfirl in the heart was only rescued by aggressive iron therapy, which overwhelmed the capacity of serum Tf to bind iron. The phenotype was ameliorated by expression of an iron uptake-incompetent Tfirl (i.e., Tf _r i ^R564A ) ₀ r administration of nicotinamide riboside, an NAD precursor. Nicotinamide riboside increased the lifespan of treated mice in the absence of iron therapy, and thus, may be a therapeutic approach for patients with heart failure complicated by iron deficiency.

6. Clauses

[00130] Clause 1. A method for treating heart failure in a subject in need thereof, the method comprising administering a therapeutically effective amount of nicotinamide riboside to the subject.

[00131] Clause 2. The method of clause 1, wherein the subject has iron deficiency.

[00132] Clause 3. The method of clause 1, wherein the subject has iron deficiency in the heart.

[00133] Clause 4. The method of clause 1, wherein the subject has iron deficiency with anemia.

[00134] Clause 5. The method of clause 1, wherein the subject has a decreased NAD/NADH ratio as compared to a NAD/NADH ratio of a subject not suffering from heart failure.

[00135] Clause 6. The method of clause 1, wherein the subject has mitochondrial dysfunction.

[00136] Clause 7. The method of clause 1, wherein the subject has reduced or dysfunctional mitophagy.

[00137] Clause 8. The method of clause 1, further comprising administering iron to the subject.

[00138] Clause 9. The method of clause 1, wherein a lifespan of the subject is extended as compared to a lifespan of a subject suffering from heart failure and not administered

nicotinamide riboside.

[00139] Clause 10. A method for treating heart failure in a subject in need thereof, the method comprising administering a therapeutically effective amount of nicotinamide riboside and a therapeutically effective amount of iron to the subject.

[00140] Clause 11. The method of clause 10, wherein the heart failure is associated with iron deficiency. [00141] Clause 12. A method for treating heart failure associated with iron deficiency in a subject in need thereof, the method comprising administering a therapeutically effective amount of nicotinamide riboside to the subject.

[00142] Clause 13. The method of clause 12, wherein the iron deficiency is in the heart of the subject.

[00143] Clause 14. The method of clause 12, wherein anemia is present with the iron deficiency.

[00144] Clause 15. The method of clause 12, wherein the subject has a decreased NAD/NADH ratio as compared to a NAD/NADH ratio of a subject not suffering from heart failure associated with iron deficiency.

[00145] Clause 16. The method of clause 12, wherein the subject has mitochondrial dysfunction.

[00146] Clause 17. The method of clause 12, wherein the subject has reduced or dysfunctional mitophagy.

[00147] Clause 18. The method of clause 12, further comprising administering iron to the subject.

[00148] Clause 19. The method of clause 12, wherein a lifespan of the subject is extended as compared to a lifespan of a subject suffering from heart failure associated with iron deficiency and not administered nicotinamide riboside.

[00149] Clause 20. A method for treating heart failure associated with iron deficiency in a subject in need thereof, the method comprising administering a therapeutically effective amount of nicotinamide riboside and a therapeutically effective amount of iron to the subject.

[00150] Clause 21. A method of identifying a subject suffering from heart failure as a candidate for treatment with nicotinamide riboside, the method comprising: (a) measuring in a sample obtained from the subject a level selected from the group consisting of: (i) a level of a ratio of NAD/NADH; (ii) a level of iron; (iii) a level of FGF21 protein; (iv) a level of

Angiopoietin-like 4 (AngPtL4) protein; and (v) any combination thereof; (b) comparing the measured level to a control level; and (c) identifying the subject as a candidate for treatment with nicotinamide riboside if (i) the measured level of the ratio of NAD/NADH is lower than the control level of the ratio of NAD/NADH; (ii) the measured level of iron is lower than the control level of iron; (iii) the measured level of FGF21 protein is higher than the control level of FGF21 protein; (iv) the measured level of AngPtL4 protein is higher than the control level of AngPtL4 protein; or (v) any combination thereof.

[00151] Clause 22. The method of clause 21, wherein the heart failure is associated with iron deficiency.

[00152] Clause 23. The method of clause 22, wherein the iron deficiency is in the heart of the subject.

[00153] Clause 24. The method of clause 22, wherein anemia is present with the iron deficiency.

[00154] Clause 25. The method of clause 21, wherein the subject has mitochondrial dysfunction.

[00155] Clause 26. The method of clause 21, wherein the subject has reduced or dysfunctional mitophagy.

[00156] Clause 27. The method of clause 21, further comprising administering a

therapeutically effective amount of nicotinamide riboside to the subject identified as a candidate for treatment with nicotinamide riboside.

[00157] Clause 28. The method of clause 27, wherein administration of nicotinamide riboside includes administration of iron to the subject identified as a candidate for treatment with nicotinamide riboside.

[00158] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the invention, which is defined solely by the appended claims and their equivalents.

[00159] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the invention, may be made without departing from the spirit and scope thereof.