Nicotinamide Riboside-Driven Modulation of SIRT3/mtROS/JNK Signaling Pathways Alleviates Myocardial Ischemia-Reperfusion Injury

Abstract

Myocardial ischemia-reperfusion (I/R) injury exacerbates cellular damage upon restoring blood flow to ischemic cardiac tissue, causing oxidative stress, inflammation, and apoptosis. This study investigates Nicotinamide Riboside (NR), a precursor of nicotinamide adenine dinucleotide (NAD⁺), for its cardioprotective effects. Administering NR to mice before I/R injury and evaluating heart function via echocardiography showed that NR significantly improved heart function, increased left ventricular ejection fraction (LVEF) and fractional shortening (FS), and reduced left ventricular end-diastolic (LVDd) and end-systolic diameters (LVSd). NR also restored E/A and E/e' ratios. It reduced cardiomyocyte apoptosis both in vivo and in vitro, inhibiting elevated caspase-3 activity and returning Bax protein levels to normal. In vitro, NR reduced the apoptotic rate in hydrogen peroxide (H2O2)-treated HL-1 cells from 30% to 10%. Mechanistically, NR modulated the SIRT3/mtROS/JNK pathway, reversing H2O2-induced SIRT3 downregulation, reducing mitochondrial reactive oxygen species (mtROS), and inhibiting JNK activation. Using SIRT3-knockout (SIRT3-KO) mice, we confirmed that NR's cardioprotective effects depend on SIRT3. Echocardiography showed that NR's benefits were abrogated in SIRT3-KO mice. In conclusion, NR provides significant cardioprotection against myocardial I/R injury by enhancing NAD+ levels and modulating the SIRT3/mtROS/JNK pathway, suggesting its potential as a novel therapeutic agent for ischemic heart diseases, meriting further clinical research.

Keywords: JNK; Nicotinamide Riboside; SIRT3; cardiac ischemia reperfusion injury.; mtROS.

Figure 1

NR Improves Heart Function After Myocardial Ischemia-Reperfusion (I/R) Injury. Heart function was evaluated using echocardiography in mice subjected to I/R injury and treated with various doses of Nicotinamide Riboside (NR). (A) Left ventricular ejection fraction (LVEF) and (B) fractional shortening (FS) were significantly reduced in the I/R group compared to the sham group, indicating impaired heart function. (C) The E/A ratio and (D) E/e' ratio, indicators of diastolic function, were also diminished in the I/R group. (E) Left ventricular end-diastolic diameter (LVDd) and (F) left ventricular end-systolic diameter (LVSd) were enlarged in response to I/R injury. NR administration significantly improved these parameters in a dose-dependent manner, suggesting its potential therapeutic benefit in mitigating I/R-induced cardiac dysfunction. *p<0.05 indicates a statistically significant difference compared to the sham group.

Figure 2

NR Reduces I/R-Mediated Cardiomyocyte Apoptosis In Vivo and In Vitro. (A) Caspase-3 activity, a marker of apoptosis, was significantly elevated in the I/R group compared to the sham group. NR treatment dose-dependently inhibited this increase, as measured by ELISA. (B) The pro-apoptotic protein Bax was upregulated in response to I/R injury but returned to physiological levels with NR treatment. (C) In vitro experiments using HL-1 cells treated with 0.3 mM hydrogen peroxide (H₂O₂) to mimic I/R injury showed that NR administration significantly reduced the apoptotic rate from approximately 33% to 8%, as determined by TUNEL staining. These findings collectively indicate that NR markedly reduces cardiomyocyte apoptosis both in vivo and in vitro. *p<0.05 indicates a statistically significant difference compared to the I/R group without NR treatment.

Figure 3

NR Regulates the SIRT3/mtROS/JNK Pathway. (A) Western blot analysis showed that SIRT3 expression was significantly downregulated following H₂O₂ treatment in HL-1 cells. NR treatment reversed this downregulation, restoring SIRT3 levels. (B) Immunofluorescence assays demonstrated a significant increase in mitochondrial reactive oxygen species (mtROS) production in response to H₂O₂, which was reduced by NR treatment. (C) ELISA results showed that H₂O₂ significantly elevated JNK activity, which was abolished by NR treatment. These results demonstrate that NR reverses the downregulation of SIRT3, preventing mtROS accumulation and JNK activation in H₂O₂-treated cardiomyocytes. *p<0.05 indicates a statistically significant difference compared to the H₂O₂-treated group without NR.

Figure 4

Cardioprotective Effects of NR Are Abrogated in SIRT3-Knockout Mice. Heart function was evaluated using echocardiography in both wild-type (WT) and SIRT3-knockout (SIRT3-KO) mice subjected to I/R injury. (A) Left ventricular ejection fraction (LVEF) and (B) fractional shortening (FS) were impaired in WT mice following I/R injury, but NR treatment improved these parameters. In SIRT3-KO mice, the cardioprotective effects of NR were abrogated, as evidenced by the lack of improvement in LVEF and FS. (C) The E/A ratio and (D) E/e' ratio, which were normalized by NR in WT mice, were not improved in SIRT3-KO mice. (E) Left ventricular end-diastolic diameter (LVDd) and (F) left ventricular end-systolic diameter (LVSd) were not improved in SIRT3-KO mice. These findings suggest that the cardioprotective effects of NR are highly dependent on the presence of SIRT3. *p<0.05 indicates a statistically significant difference compared to the WT I/R group without NR treatment.