Sexually dimorphic effects of SARM1 deletion on cardiac NAD+ metabolism and function.

Nicotinamide adenine dinucleotide (NAD+) decline is repeatedly observed in heart disease and its risk factors. Although strategies promoting NAD+ synthesis to elevate NAD+ levels improve cardiac function, whether inhibition of NAD+ consumption can be therapeutic is less investigated. In this study, we examined the role of sterile-α and TIR motif containing 1 (SARM1) NAD+ hydrolase in mouse hearts, using global SARM1-knockout mice (KO). Cardiac function was assessed by echocardiography in male and female KO mice and wild-type (WT) controls. Hearts were collected for biochemical, histological, and molecular analyses. We found that the cardiac NAD+ pool was elevated in female KO mice, but only trended to increase in male KO mice. SARM1 deletion induced changes to a greater number of NAD+ metabolism transcripts in male mice than in female mice. Body weights, cardiac systolic and diastolic function, and geometry showed no changes in both male and female KO mice compared with WT counterparts. Male KO mice showed a small, but significant, elevation in cardiac collagen levels compared with WT counterparts, but no difference in collagen levels was detected in female mice. The increased collagen levels were associated with greater number of altered profibrotic and senescence-associated inflammatory genes in male KO mice, but not in female KO mice.NEW & NOTEWORTHY We examined the effects of SARM1 deletion on NAD+ pool, transcripts of NAD+ metabolism, and fibrotic pathway for the first time in mouse hearts. We observed the sexually dimorphic effects of SARM1 deletion. How these sex-dependent effects influence the outcomes of SARM1 deficiency in male and female mice in responses to cardiac stresses warrant further investigation. The elevation of cardiac NAD+ pool by SARM1 deletion provides evidence that targeting SARM1 may reverse disease-related NAD+ decline.

Chelonian perivitelline membrane-bound sperm detection: A new breeding management tool.

Perivitelline membrane (PVM)-bound sperm detection has recently been incorporated into avian breeding programs to assess egg fertility, confirm successful copulation, and to evaluate male reproductive status and pair compatibility. Due to the similarities between avian and chelonian egg structure and development, and because fertility determination in chelonian eggs lacking embryonic growth is equally challenging, PVM-bound sperm detection may also be a promising tool for the reproductive management of turtles and tortoises. This study is the first to successfully demonstrate the use of PVM-bound sperm detection in chelonian eggs. Recovered membranes were stained with Hoechst 33342 and examined for sperm presence using fluorescence microscopy. Sperm were positively identified for up to 206 days post-oviposition, following storage, diapause, and/or incubation, in 52 opportunistically collected eggs representing 12 species. However, advanced microbial infection frequently hindered the ability to detect membrane-bound sperm. Fertile Centrochelys sulcata, Manouria emys, and Stigmochelys pardalis eggs were used to evaluate the impact of incubation and storage on the ability to detect sperm. Storage at -20°C or in formalin were found to be the best methods for egg preservation prior to sperm detection. Additionally, sperm-derived mtDNA was isolated and PCR amplified from Astrochelys radiata, C. sulcata, and S. pardalis eggs. PVM-bound sperm detection has the potential to substantially improve studies of artificial incubation and sperm storage, and could be used to evaluate the success of artificial insemination in chelonian species. Mitochondrial DNA from PVM-bound sperm has applications for parentage analysis, the study of sperm competition, and potentially species identification.

Genetic architecture of heart mitochondrial proteome influencing cardiac hypertrophy.

Mitochondria play an important role in both normal heart function and disease etiology. We report analysis of common genetic variations contributing to mitochondrial and heart functions using an integrative proteomics approach in a panel of inbred mouse strains called the Hybrid Mouse Diversity Panel (HMDP). We performed a whole heart proteome study in the HMDP (72 strains, n=2-3 mice) and retrieved 848 mitochondrial proteins (quantified in ≥50 strains). High-resolution association mapping on their relative abundance levels revealed three trans-acting genetic loci on chromosomes (chr) 7, 13 and 17 that regulate distinct classes of mitochondrial proteins as well as cardiac hypertrophy. DAVID enrichment analyses of genes regulated by each of the loci revealed that the chr13 locus was highly enriched for complex-I proteins (24 proteins, P=2.2E-61), the chr17 locus for mitochondrial ribonucleoprotein complex (17 proteins, P=3.1E-25) and the chr7 locus for ubiquinone biosynthesis (3 proteins, P=6.9E-05). Follow-up high resolution regional mapping identified NDUFS4, LRPPRC and COQ7 as the candidate genes for chr13, chr17 and chr7 loci, respectively, and both experimental and statistical analyses supported their causal roles. Furthermore, a large cohort of Diversity Outbred mice was used to corroborate Lrpprc gene as a driver of mitochondrial DNA (mtDNA)-encoded gene regulation, and to show that the chr17 locus is specific to heart. Variations in all three loci were associated with heart mass in at least one of two independent heart stress models, namely, isoproterenol-induced heart failure and diet-induced obesity. These findings suggest that common variations in certain mitochondrial proteins can act in trans to influence tissue-specific mitochondrial functions and contribute to heart hypertrophy, elucidating mechanisms that may underlie genetic susceptibility to heart failure in human populations.

NAD+ Redox Imbalance in the Heart Exacerbates Diabetic Cardiomyopathy.

BACKGROUND: Diabetes is a risk factor for heart failure and promotes cardiac dysfunction. Diabetic tissues are associated with nicotinamide adenine dinucleotide (NAD+) redox imbalance; however, the hypothesis that NAD+ redox imbalance causes diabetic cardiomyopathy has not been tested. This investigation used mouse models with altered NAD+ redox balance to test this hypothesis. METHODS: Diabetic stress was induced in mice by streptozotocin. Cardiac function was measured by echocardiography. Heart and plasma samples were collected for biochemical, histological, and molecular analyses. Two mouse models with altered NAD+ redox states (1, Ndufs4 [NADH:ubiquinone oxidoreductase subunit S4] knockout, cKO, and 2, NAMPT [nicotinamide phosphoribosyltranferase] transgenic mice, NMAPT) were used. RESULTS: Diabetic stress caused cardiac dysfunction and lowered NAD+/NADH ratio (oxidized/reduced ratio of nicotinamide adenine dinucleotide) in wild-type mice. Mice with lowered cardiac NAD+/NADH ratio without baseline dysfunction, cKO mice, were challenged with chronic diabetic stress. NAD+ redox imbalance in cKO hearts exacerbated systolic (fractional shortening: 27.6% versus 36.9% at 4 weeks, male cohort P<0.05), and diastolic dysfunction (early-to-late ratio of peak diastolic velocity: 0.99 versus 1.20, P<0.05) of diabetic mice in both sexes. Collagen levels and transcripts of fibrosis and extracellular matrix-dependent pathways did not show changes in diabetic cKO hearts, suggesting that the exacerbated cardiac dysfunction was due to cardiomyocyte dysfunction. NAD+ redox imbalance promoted superoxide dismutase 2 acetylation, protein oxidation, troponin I S150 phosphorylation, and impaired energetics in diabetic cKO hearts. Importantly, elevation of cardiac NAD+ levels by NAMPT normalized NAD+ redox balance, alleviated cardiac dysfunction (fractional shortening: 40.2% versus 24.8% in cKO:NAMPT versus cKO, P<0.05; early-to-late ratio of peak diastolic velocity: 1.32 versus 1.04, P<0.05), and reversed pathogenic mechanisms in diabetic mice. CONCLUSIONS: Our results show that NAD+ redox imbalance to regulate acetylation and phosphorylation is a critical mediator of the progression of diabetic cardiomyopathy and suggest the therapeutic potential for diabetic cardiomyopathy by harnessing NAD+ metabolism.