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.

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.

AbstractBackgroundDiabetes is a risk factor of heart failure, and chronic diabetic stress leads to cardiac dysfunction. Diabetic tissues are associated with NAD+ redox imbalance; however, the hypothesis that NAD+ redox imbalance leads to dysfunction of diabetic hearts has not been tested. In this study, we employed mouse models with altered NAD+ redox balance to test the hypothesis and to characterize the underlying mechanism.Methods and ResultsType 1 diabetes was induced in C57/BL6 mice by streptozotocin injections, and diabetic cardiomyopathy (DCM) was allowed to develop for 16-weeks. Chronic diabetic stress led to cardiac dysfunction and lowered NAD+/NADH ratio. The same diabetogenic regimen was administered to cardiac-specific Ndufs4-KO mice (cKO), a model of lowered cardiac NAD+/NADH ratio without overt baseline dysfunction. Cardiac NAD+ redox imbalance in cKO hearts exacerbated systolic and diastolic dysfunction of diabetic mice in both sexes. Collagen levels and transcript analyses of fibrosis and extracellular matrix-dependent pathways showed no change in diabetic cKO hearts, suggesting that the exacerbated cardiac dysfunction was likely due to cardiomyocyte dysfunction. We found that cardiac NAD+ redox imbalance promoted SOD2 acetylation and protein oxidation, and regulated TnI phosphorylation in diabetic cKO hearts. Elevation of cardiac NAD+ levels by NAMPT over-expression mitigated NAD+ redox imbalance in diabetic cKO mice, reversed SOD2 acetylation and TnI phosphorylation, and alleviated cardiac dysfunction.ConclusionOur results show that NAD+ redox imbalance to regulate protein acetylation and phosphorylation is a critical mediator of the progression of DCM, and suggest the therapeutic potential of harnessing NAD+ metabolism in DCM.

NAD+ decline is repeatedly observed in heart disease and its risk factors. While 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 SARM1 (Sterile alpha and TIR motif containing 1) NAD+ hydrolase in mouse heart, using a 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 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 to WT counterparts. Male KO mice showed a small, but significant elevation in cardiac collagen levels compared to 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.

Our lab studies how natural genetic variations affect common diseases using a mouse population called hybrid mouse diversity panel (HMDP). In this study, we have explored the genetic regulation of mitochondrial pathways and their contribution to heart function using an integrative proteomics approach. We first performed a whole heart proteomic analysis in the HMDP (72 strains, n=2-3 mice) and surveyed mitochondrial localization using MitoCarta2.0. We retrieved 840 of these proteins (quantified in ≥50 strains) and performed high-resolution association mapping on their respective abundance levels to the HMDP genotypes. Our analyses identified three genetic loci, located on chromosome (chr) 7, chr13 and chr17, that control distinct classes of mitochondrial proteins as well as heart hypertrophy. Follow-up high resolution regional mapping identified NDUFS4, LRPPRC and COQ7 as the candidate genes for chr13, chr17 and chr7 loci, respectively. All three are associated with heart mass in two independent heart stress models, namely, isoproterenol (ISO)-induced heart failure and diet-induced obesity (DIO) models. Next, to identify the aspects of mitochondrial metabolism regulated by these loci, we constructed co-expression protein networks using weighted gene co-expression network analysis (WGCNA) and identified five modules. Eigengenes, representing the first principal component of two of these modules (Brown and Green), mapped to the same regions as the chr13 and chr17 loci, respectively. DAVID enrichment analyses revealed that the Brown module (72 proteins, 96% overlap with chr13) was highly enriched for complex-I proteins (35 proteins, P = 8.8E-74) and the Green module (44 proteins, 73% overlap with chr17) for mitochondrial ribosomal proteins (25 proteins, P = 1.3E-53). The proteins in the chr7 locus were found primarily in the Turquoise module (393 proteins, 81% overlap) but this module was not enriched for any single mitochondrial protein complex. In summary, we now report the identification of three genetic loci that control distinct classes of mitochondrial proteins as well as heart hypertrophy. Our results provide strong support for a role of the mitochondrial proteome in heart pathophysiology.