3.1. Hyperacetylation of mitochondrial proteins in KLF4-deficient hearts
We previously reported that cardiac KLF4-deficient mice (CM- K4KO) developed acute heart failure and death in response to pressure overload induced by transverse aortic constriction (TAC) due to dis- rupted mitochondrial homeostasis [18,20]. Consistently, the tran- scriptomic study revealed ∼4000 differentially expressed genes (FDR < 0.05) between the MHC-Cre and CM-K4KO groups after 3-day of TAC, underscoring the sharp difference observed in cardiac function upon stress . However, at baseline, only ∼80 genes were identified as being differentially expressed between the MHC-Cre and CM-K4KO groups suggesting minimal changes at the transcriptional level . However, we did observe reduced cardiac mitochondrial function at baseline despite overall heart function is normal in those young adult mice (age: 8–16 weeks) . Furthermore, these mutant mice gradu- ally developed age-related cardiomyopathy coupled with mitochondrial
degeneration . These data suggest that posttranslational mechan- isms might contribute to the mitochondrial defects.
We first analyzed the mitochondrial electron transport chain (ETC) complexes by Blue-Native-PAGE (BN-PAGE) and found no difference in ETC complex assembly between the CM-K4KO and MHC-Cre groups at baseline (Fig. 1A). As protein acetylation has been shown to negatively affect mitochondrial function, we subjected cardiac mitochondrial proteins to Western blot analysis for acetyl-Lysine to assess overall protein acetylation levels in the mitochondria. As shown in Fig. 1B, there was marked increase of protein acetylation in the CM-K4KO group. Pressure overload induced by transverse aortic constriction (TAC) further enhanced mitochondrial protein acetylation and the same hyperacetylation in CM-K4KO group persisted even after TAC (Fig. 1C). Such hyperacetylation of mitochondrial proteins was also associated with TAC-induced heart failure in C57BL6 WT mice (Fig. 1D), sug- gesting a plausible relationship between hyperacetylation of cardiac mitochondrial proteins and cardiac dysfunction.
To gain a further understanding of these hyper-acetylated proteins, we immunoprecipitated all acetylated mitochondrial proteins by the anti-acetyl-Lysine antibody and probed for Superoxide dismutase 2 (SOD2), Cyclophilin D (CypD) and long chain Acyl-CoA dehydrogenase (LCAD), all of which are known Sirt3 targets and can be acetylated under certain conditions [12,21–23]. As shown in Fig. 1E, all three acetylated proteins were enriched in the CM-K4KO group. Previous studies have reported that hyperacetylation of these proteins are asso- ciated with mitochondrial dysfunction and heart failure [12,21–23]. As such, our data suggested that mitochondria in the KLF4-deficient car- diomyocytes were likely predisposed to hyperacetylation-related de- fects. Previously we showed impaired mitochondrial metabolic function in the KLF4-deficient heart , which can be partly attributed to protein hyperacetylation.
Collectively, these data demonstrate that cardiac KLF4-deficiency led to hyperacetylation of mitochondrial proteins, likely due to reduced deacetylation function. Such posttranslational modification of mi- tochondrial proteins can profoundly impair mitochondrial metabolic function at baseline, which may predispose the CM-K4KO hearts vul- nerable to stress [18,20].
Fig. 5. Administration of NMN protected cardiac mitochondria from TAC-induced damage. (A) EM images from PBS (vehicle) treated hearts after 5 days of TAC. (B) EM images from NMN (500 mg/kg/day) treated hearts after 5 days of TAC. Scale bar: 1 um. Arrows indicate damaged or abnormal mitochondria. Each image was from individual animal but different areas were chosen to display different phenotype. n = 3 in each group.
3.2. Reduction of Sirt3, NAD+ and NAMPT in the KLF4-deficient heart
Mitochondrial protein acetylation is mainly regulated mainly by micotinamide adenine dinucleotide (NAD+) and the Sirtuin family of NAD+-dependent deacetylases. Among all known mammalian Sirtuin genes, we found the mRNA levels of Sirt3 and Sirt5 were reduced in CM-K4KO heart (Fig. 2A). Of note, Sirt3 is the key deacetylase in the mitochondrial matrix while Sirt5 is more of a deacylase rather than a deacetylase . Hence, we focused on Sirt3 and found its protein le- vels were also reduced in the CM-K4KO mitochondria (Fig. 2B). Al- though it is hard to determine if such subtle decrease of Sirt3 protein is rate-limiting, undoubtably Sirt3 insufficiency can contribute to mi- tochondrial protein hyperacetylation .
Since NAD+ is critical for protein acetylation as well as mitochon- drial respiration function, we next sought to determine the NAD+ levels in cardiac tissue. NAD+ levels were reduced in CM-K4KO hearts and pressure overload further affect myocardial NAD+ levels (Fig. 2C). Not surprisingly, TAC reduced cardiac NAD+ levels to much lower levels in the CM-K4KO group (Fig. 2C). NAD+ biosynthesis relies on two path- ways, namely de novo and salvage pathways (Fig. 2D). Because adult heart does not express TDO (tryptophan 2,3-dioxygenase) or IDO (In- doleamine 2,3-dioxygenase), two rate-limiting enzymes in the de novo pathway, nearly all cardiac NAD+ is generated from the salvage pathway . Nicotinamide phosphoribosyltransferase (NAMPT), the key enzyme in NAD+ salvage pathway, was significantly lower in the
CM-K4KO heart and it went further down after TAC (Fig. 2E). These data strongly suggested a NAD+ deficient stage in the CM-K4KO heart, especially after the onset of pressure overload.
The combination of reduced expression of Sirt3 and low NAD+ concentration could result in overall deacetylase activity deficiency in the mitochondria leading to mitochondrial protein hyperacetylation. Moreover, NAD+ is a key coenzyme of the tricarboxylic acid (TCA) cycle and fatty acid β-oxidation, a depleted NAD+ pool would also impair mitochondrial metabolism.
3.3. Administration of NMN corrects mitochondrial acetylation and rescues the heart
Given the mitochondrial hyperacetylation and NAD+ deficiency observed in the CM-K4KO hearts, we asked if NAD+ repletion could be beneficial to these animals. To bypass the NAMPT defect, we chose to use Nicotinamide Mononucleotide (NMN) as the exogenous NAD+ precursor (Fig. 2D). As expected, NMN administration increased cardiac tissue NAD+ level (Fig. 3A). Further, NMN normalized the mitochon- drial protein acetylation levels in the hearts (Fig. 3B). Strikingly, ad- ministration of NMN at 500 mg/kg/day starting from one day before TAC profoundly preserved the cardiac contractile function and com- pletely rescued the heart failure phenotype in the CM-K4KO group (Fig. 3C). NMN treatment did not affect the expression of hypertrophic genes (i.e. ANF, β-MHC) but blunted the induction of inflammatory genes (i.e. IL1β, CCL2 and IL6) in CM-K4KO hearts (Fig. 3D), indicating that administration of NMN maintained a normal hypertrophic re- sponse to pressure overload but reduced cardiac injury. Due to high mortality rate of CM-K4KO mice after TAC , the phenotypical comparison between NMN and PBS (Vehicle) administration were performed within 5 days post-TAC but the CM-K4KO animals with T- AC + NMN administration exhibited 100% survival rate during the course of study (data not shown).
Fig. 6. Administration of NMN reduced TAC-induced ROS generation in KLF4-deficient hearts. (A) Representative images showing myocardial DHE staining. (B) ROS level was calculated as DHE positive cells per field. TAC and NMN administra- tion: 5 days. n = 3–5 animals in each group. *p < 0.05.