Heart failure (HF) poses a serious threat to human health and is characterized by high morbidity, high mortality, a high rehospitalization rate, and many high-risk groups. The American Heart Association/American College of Cardiology guidelines define HF as “a complex clinical syndrome caused by any structural or functional heart disease that affects the ability of the ventricles to fill and shoot blood” [1]. Benjamin et al. [2] reported that patients hospitalized for HF are at an increased risk of HF rehospitalization and cardiovascular death. One in eight deaths is due to HF, and the mortality rate within 5 years of diagnosis is as high as 50%, exceeding that of some malignancies [2].

Despite the increased number of drugs used to treat HF, the fatality rate remains high. The 2021 European Society of Cardiology guidelines recommend a combination of angiotensin-converting enzyme inhibitor (ACEI)/angiotensin-receptor neprilysin inhibitor, $\beta$-blockers, and aldosterone receptor antagonists (MRAs) as the primary treatment for chronic HF [3]. Both the previous “golden triangle” ($\beta$-blocker, ACEI/angiotensin II receptor blocker [ARB], and MRA) and the current “five golden flowers” ($\beta$-blocker, ACEI/ARB, MRA, and sodium-dependent glucose transporter 2 inhibitors) have limitations in clinical treatment [4, 5, 6]. Current drug therapy still cannot meet the needs of HF management, making it necessary to develop new treatment ideas and explore new treatment strategies.

Nicotinamide adenine dinucleotide (NAD⁺) is an important cofactor and a key metabolic enzyme substrate involved in redox reactions in the mitochondria, which is greatly significant in maintaining the balance of NAD⁺ in vivo for normal human metabolism [7]. Several studies have shown that NAD⁺ can inhibit inflammation and oxidative stress injury in endothelial cells, reduce apoptosis in microvascular endothelial cells, promote microangiogenesis, and improve microvascular injury caused by coronary microcirculation and myocardial ischemia-reperfusion [8]. Studies have also shown that the NAD⁺ concentration in the blood of patients with HF is significantly lower than in healthy people, and with an increase in age, NAD⁺ also has a trend of gradually decreasing [7, 9]. Therefore, stabilizing intracellular NAD⁺ levels through exogenous NAD⁺ supplementation is expected to be a therapeutic strategy for improving cardiac bioenergetics and function.

HF is the leading cause of hospitalization among older populations worldwide, with a high mortality rate and impact on the quality of life of patients [10, 11]. With the development of medical technology, various drugs have emerged to treat HF. However, the effectiveness of these drugs remains unsatisfactory. HF remains an intractable disease, with an increased burden of hospitalization and a continuous loss of health care expenditure [12]. Therefore, it is necessary to identify alternative and complementary treatment options. Thus, we sought to evaluate whether NAD⁺ therapy has some clinical efficacy in patients with HF.

NAD⁺ is widely present in the mitochondria of myocardial tissue, and NAD⁺ activates the deacetylation activity of sirtuins, regulates the activity of numerous aging-related transcription factors, and intervenes in aging and aging-related diseases [9, 13, 14]. Several studies have shown that NAD⁺ has beneficial effects on cardiovascular diseases by regulating metabolism, maintaining redox homeostasis, and modulating immune responses [15, 16, 17]. Studies have found that NAD⁺ levels are significantly reduced in patients with HF [18, 19]. In a rat model of myocardial infarction, compared with the LCZ696-positive drug control group, using NAD⁺ improved some cardiac function and hemodynamic indexes and could further increase the left ventricular stroke output and systolic and diastolic blood pressure [20]. In the present clinical trial, we tested whether using exogenous NAD⁺ supplementation as a new adjuvant treatment for HF is possible.

NT-proBNP and LVEF are the most widely used laboratory indices for evaluating HF severity and prognosis. In our clinical trial, NT-proBNP levels improved in both groups, possibly because patients in both groups were rigorously treated with anti-HF drugs. However, the improvement was more obvious in the NAD⁺ group than in the saline control group, which better reflects that the therapeutic effect of NAD⁺ in patients with HF is independent of conventional medication for HF. Although the NT-proBNP level in the NAD⁺ group improved from baseline, no statistically significant difference was found over time. This finding may be due to the large dispersion of the data, short treatment course, and small sample size; similarly, due to short-term drug use, changes in the structure and function of the heart cannot be obvious over a short time. Moreover, the measurement of LVEF is subjective to a certain extent and is related to the experience of the tester. LVEF in the NAD⁺ group was improved, with no statistical difference between the groups. Studies have found that women may have lower NAD⁺ concentrations and benefit most from improved heart function [21, 22]. In our study, there was no statistical difference in gender between the two groups of patients, and whether women can benefit more from NAD⁺ treatment will be our major direction in the future.

Oxidative stress plays an important role in HF occurrence and progression [23]. The sirtuin family is also linked to several antioxidant and oxidative stress-related processes and functions [24]. Sirtuins are a family of seven enzymes (sirtuin1–7) involved in regulating many metabolic processes [25]. Sirtuin agonists are more convincing than existing deacetylase inhibitors for the treatment of cardiovascular diseases in terms of safety and efficacy and may have clinical value for treating multiple types of cardiovascular diseases [26]. Increasing the NAD⁺ level in vivo can activate sirtuins, which can significantly inhibit myocardial hyperacetylation and improve myocardial mitochondrial function [27]. Clemency et al. [28] found that sodium-glucose cotransporter 2 (SGL-2) inhibitor could inhibit oxidative stress by increasing the expression of SIRT1 and SIRT3 and decreasing the expression of SIRT6, thereby alleviating myocardial injury. In our clinical trial, the SIRT1, SIRT3, and SIRT6 levels increased at the 2-week follow-up after medication, but over time, the concentration of exogenous supplemental NAD⁺ gradually decreased in vivo and showed a trend of gradual decrease in the last two follow-up visits, which is also consistent with the metabolic process of intravenous drug use in the body. Furthermore, there was an interaction between SIRT1 in the saline control group and the NAD⁺ group, which confirmed that NAD⁺ plays a critical role in anti-oxidative stress. We observed that ROS levels increased in the NAD⁺ group at the 2-week follow-up visit, possibly due to the negative feedback reaction of inflammation caused by the strong antioxidant effect in the short term. However, those levels improved at later visits.

Increased levels of ET, the most potent vasoconstrictor, are produced by the pro-peptide precursor, large ET, through ET convertase [29]. ET in the peripheral blood of patients with HF can predict poor prognosis [30, 31, 32]. In this clinical trial, there were significant differences in ET levels between the saline control and NAD⁺ groups, which confirmed that NAD⁺ plays a more critical role in anti-endothelial injury.

Although there was no difference in the incidence of composite endpoint events (including all-cause death and readmission due to HF) during the 1-year follow-up period in this study, there was still a significant difference in mortality between the two groups. The survival rate of NAD⁺ was higher, and most patients who died were in the saline control group, usually 2–4 months after treatment. These results suggest that the use of NAD⁺ may delay the progression of HF and reduce short-term mortality. The reason why no statistical difference was found in the various clinical endpoints in this study may be related to the small number of study cases, the short duration of NAD⁺ administration, and the short follow-up time. Nevertheless, this study has expanded our ideas for exploring new treatments for patients with HF and confirmed their therapeutic effect on patients with HF based on molecular biology and echocardiography evaluation indicators. We also expect this study’s results to guide follow-up national multicenter, large-sample, prospective, randomized, double-blind controlled studies. We further confirmed the therapeutic effects of NAD⁺ in the HF population.

According to the product instructions, NAD⁺ occasionally has side effects such as dry mouth, nausea, dizziness, and palpitations. However, in this clinical trial, patients had no obvious complaints after 7 days of intravenously administering NAD⁺, but the long-term effect or safety of treatment still needs to be observed over a longer study period. In addition, the follow-up of participants after completing this clinical trial may be limited, making it difficult to assess long-term outcomes and treatment safety.

Our study consisted of a small sample size from a single center, potentially limiting the generalizability of our findings to a broader population of HF patients. Future multicenter studies covering cohorts with different demographic and clinical characteristics would help validate our findings and enhance external validity. In addition, we did not perform cardiovascular magnetic resonance to assess patients with HF more comprehensively. We did not discuss the pharmacological background of HF patients further, and our future research direction will be to focus on their pharmacological background.

Among patients with HF, those injected with NAD⁺ for 7 days may benefit more from improved cardiac function, levels of anti-oxidative stress, and endothelial injury than those receiving saline. A prospective observational study will determine the efficacy and safety of NAD⁺, a promising adjuvant therapy for patients with HF.

The data underlying this study are not publicly available as they include patient-level data, but are available from the corresponding author on reasonable request.

FW and ZWP designed the research study. XYM and YG performed the research. MD analyzed the data. WY prepared figures and drafted the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Beijing hospital (Date18/3/2021/No2021BJYYEC-034-02). Informed consent was obtained from all individual participants included in the study.

We thank Knature Company (Hefei Knature Bio-pharm Co., Ltd,) for providing freeze-dry powder injector NAD⁺.

This work was supported by National High Level Hospital Clinical Research Funding (BJ-2023-107), National High Level Hospital Clinical Research Funding (BJ-2022-117), and Knature Company (Grant number 202101-05-02).

The authors declare no conflict of interest. Knature Company (Hefei Knature Biopharm Co., Ltd,) was not involved in this study and the authors declare that the company has no conflict of interest.