Nicotinamide Riboside Preserves Cardiac Function in a Mouse Model of Dilated Cardiomyopathy
Authors: István Baczkó
Jocelyne Blanc
Cynthia Tannous
Nicolas Diguet
Boris Manoury
Nathalie Mougenot
Anne Gouge
Gareth G Lavery
Robin Deloux
Zhenlin Li
Charles Brenner
Marie Breton
Anne Garnier
Jérôme Piquereau
Samuel A J Trammell
Jean-François Decaux
Mélanie Gressette
Joffrey Zoll
Mathias Mericskay
Journal: Circulation (2018)
Discipline: CLINICAL MEDICINE
Keywords: NAD; acetyl coenzyme A; cardiomyopathy, dilated; energy metabolism; glycolysis
Affiliations: Sorbonne Universités, Université Pierre et Marie Curie Paris 6, Department of Biology of Adaptation and Ageing, CNRS UMR8256, INSERM U1164, Institute of Biology Paris-Seine, DHU FAST, France (N.D., C.T., R.D., A. Gouge, J.B., J.-F.D., Z.L.)., Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City (S.A.J.T., C.B.)., Signalling and Cardiovascular Pathophysiology, UMR-S 1180, University Paris-Sud, INSERM, Université Paris- Saclay, Châtenay-Malabry, France (C.T., R.D., J.P., M.G., B.M., M.B., A. Garnier, M.M.)., Sorbonne Universités, Université Pierre et Marie Curie Paris 6, Plateforme PECMV, UMS28, Paris, France (N.M.)., Institute of Metabolism and Systems Research, University of Birmingham, United Kingdom (G.G.L.)., Department of Pharmacology and Pharmacotherapy, University of Szeged, Hungary (I.B.)., Physiology Department, Faculty of Medicine and EA3072, Université de Strasbourg, France (J.Z.)., Signalling and Cardiovascular Pathophysiology, UMR-S 1180, University Paris-Sud, INSERM, Université Paris- Saclay, Châtenay-Malabry, France (C.T., R.D., J.P., M.G., B.M., M.B., A. Garnier, M.M.). .

Abstract:  Background: Myocardial metabolic impairment is a major feature in chronic heart failure. As the major coenzyme in fuel oxidation and oxidative phosphorylation and a substrate for enzymes signaling energy stress and oxidative stress response, nicotinamide adenine dinucleotide (NAD+) is emerging as a metabolic target in a number of diseases including heart failure. Little is known on the mechanisms regulating homeostasis of NAD+ in the failing heart. Methods: To explore possible alterations of NAD+ homeostasis in the failing heart, we quantified the expression of NAD+ biosynthetic enzymes in the human failing heart and in the heart of a mouse model of dilated cardiomyopathy (DCM) triggered by Serum Response Factor transcription factor depletion in the heart (SRFHKO) or of cardiac hypertrophy triggered by transverse aorta constriction. We studied the impact of NAD+ precursor supplementation on cardiac function in both mouse models. Results: We observed a 30% loss in levels of NAD+ in the murine failing heart of both DCM and transverse aorta constriction mice that was accompanied by a decrease in expression of the nicotinamide phosphoribosyltransferase enzyme that recycles the nicotinamide precursor, whereas the nicotinamide riboside kinase 2 (NMRK2) that phosphorylates the nicotinamide riboside precursor is increased, to a higher level in the DCM (40-fold) than in transverse aorta constriction (4-fold). This shift was also observed in human failing heart biopsies in comparison with nonfailing controls. We show that the Nmrk2 gene is an AMP-activated protein kinase and peroxisome proliferator-activated receptor α responsive gene that is activated by energy stress and NAD+ depletion in isolated rat cardiomyocytes. Nicotinamide riboside efficiently rescues NAD+ synthesis in response to FK866-mediated inhibition of nicotinamide phosphoribosyltransferase and stimulates glycolysis in cardiomyocytes. Accordingly, we show that nicotinamide riboside supplementation in food attenuates the development of heart failure in mice, more robustly in DCM, and partially after transverse aorta constriction, by stabilizing myocardial NAD+ levels in the failing heart. Nicotinamide riboside treatment also robustly increases the myocardial levels of 3 metabolites, nicotinic acid adenine dinucleotide, methylnicotinamide, and N1-methyl-4-pyridone-5-carboxamide, that can be used as validation biomarkers for the treatment. Conclusions: The data show that nicotinamide riboside, the most energy-efficient among NAD precursors, could be useful for treatment of heart failure, notably in the context of DCM, a disease with few therapeutic options. 

Retracted: No

PubPeer comments:
 Figure 1. Some cells visible in panel B (QBMR treated rat) appear to also be visible in panel C (estradiol valerate treated rat). Boxes of the same color focus attention to some of those cells. This appears to be very relevant to the statement that  both QBMR and EV can restore the estrous cycle of OVX rats . ![file] , 
 Dear Dr. Elisabeth M Bikuff0c Thanks for your concern. We paid high attention on your questions that u201csome panels with repetitive elementsu201d in our articles. After checking these figures you mentioned carefully, we think your comments are inappropriate and the detailed explanations are as below: Based on our experiment, when the mice enter the estrous stage, the epithelial cells of vagina become keratinized and look very similar. Although these cells seem very similar in appearance, the numbers and distributions of nuclei are actually different, some are significant while some may be not. For example, the keratinocytes in the green frame of figure B (QBMR treated rat) contains 5 small nuclei, while the keratinocytes in the green frame of figure C (estradiol valerate treated rat) only 3 bigger nuclei (please see the parts highlighted in red arrow). Meanwhile, the shape of exfoliated cell is different in the pink frame and there are also some differences in the other two frames. Besides, the fillings of keratinocytes clusters between the panels are also obviously different, for example, there is one obvious keratinocytes cluster among the four frames in panel B(QBMR treated rat) but among the four frames in panel C (estradiol valerate treated rat) no keratinocytes cluster is observed, which indicates the different Microscopic fields of vision. Yours sincerely, Na LIN Ying Xu ![file] , 
 Dear authors, thank you for your reply, which does not make scientific sense. Although you correctly pointed put that some groups of cells are different between these 2 panels, I would like to focus attention back to the cells that look remarkably similar. The vaginal cells of 2 different rats are not expected to look this similar. What is your explanation for what happened here? How could the marked areas look so similar? , 
 Dear Dr. Elisabeth M Bikuff0c Based on our observations, almost all the epithelial cells of vagina of the rats at the estrous stage are keratinized cells which are rhombus with certain similarities in appearance indeed but the elements inside are actually very different as shown in the former pictures. As to how much similarities of these rhombus keratinized cells will be among the rats, we did not make any statistics and research on it yet, because our topic mainly focused on whether the rats enter the estrous stage after the drug treatment. Thanks for your kind understandings and any question, please contact us freely. Yours sincerely, Ying Xu , 
 #4 Dear Ying Xu, I donu2019t think you have taken the question raised by Dr. Bik seriously. It is almost impossible for cells to be similar to this level unless these mice are replicas of machines. Please also explain the similar background around the cells marked by orange boxes. ![file] , 
 #3 ![file] , 
 #4 This Figure 1 contains 2 images of vaginal epithelial cell smear. Based on my understanding and also from doi:10.3791 4389 (very informative JOVE protocol with video), the smear slides are usually prepared by the following steps: 1. wash rat s vagina using small amount of ddH2O to obtain vaginal epithelial cells in fluid 2.  Place the fluid on glass slide, and allow the smear to completely dry at room temperature 3. stain the dried smear with proper dye 4. observe under microscope to obtain the images like those in your Figure 1. Cells in the suspension obtained in steps 1 2, in my opinion, would engage in a Brownian motion which is random, until the smear dried enough to restrict any further motion of the cells, in other words, the cells location on the slide are fixed. In this sense, no any 2 smears should look so much alike, even if they might be from the same rat or even further, from the same step 1 wash but was placed onto the slide into 2 drops to create 2 smears in step 2. The  fact that 2 smear samples in your Figure 1 ended up with such almost identical patterns is a mystery to me, and probably to many reading your paper. As you said that you had observed many times this phenomenon, I d be very interested in encouraging you write it up how you create these nearly identical smears because this method might serve the purpose of creating ideal control samples for many researchers. , 
 Here I have black white reversed (c), and superimposed it on (b) with 50% transparency so that areas of image cancel out if they are present in both. ![file] Numerous keratinised epithelial cells in these two independently-obtained smears have identical outlines, backgrounds, internal details, orientations and locations relative to one another. Here is another attempt to visualise where the two images are the same, and where they differ: ![file] , 
 Thanks for your suggestions. The vaginal smear method to examine vagina cells is classical to study estrogen like activity and is simple and easy to prepare, which was recorded in the literature books of pharmacological experimental methodology and estrogen activity research references. The general steps to prepare the smear are as below: 1.twashed rat s vagina using small amount of physiological saline to obtain vaginal epithelial and dried. 2.tFixed the vaginal lavage with 95% ethanol for 10 min. 3.tStained vaginal epidermal cells with Methylene Blue for 10 min. 4.tObserved under microscopy the keratinized vaginal cells which indicates the estrus of rats. Thanks Na Lin Ying Xu , 
 #9 The question is how you create these nearly identical smears. , 
 Dear Dr. Lin and Dr. Xu, Biological tissues and cells might look similar to each other, but the chance that two cells look identical is very, very small. The chance that multiple cells in two different samples look identical to each other is even smaller. Could you please provide us with a satisfactory response to explain this? We are not particularly interested in the protocol, but in an explanation for the similarities between the 2 samples. I am looking forward to hear your reply. , 
 #9 Drs. Lin and Xu Guess you were replying specifically to my (#7) comment requesting a protocol. But as #10 explained, I was asking for your explanation on how you were able to create multiple cases where the cells and the arrangement of the cells are highly identical. , 
 Thanks for your constant concerns. Currently what we can tell is that the cells are not identical from the difference elements inside shown in the pictures as we discussed. Your concerns are interesting to us too, the chance for such similarities of cells in appearance may be less as you insisted but it does happen in practice. As to why the similarity of cells in this picture is remarkable and how it happens, frankly speaking, currently we have no knowledge of it and also donu2019t know how to reappear it because our experiment purpose did not focus on the details of similarities but to study the estrous of rats. Therefore, your kind understandings on this concern are really appreciated and we will take your concerns into consideration in our future research. Thanks Na Lin and Ying Xu ,