Elevated cardiac hemoglobin expression is associated with a pro-oxidative and inflammatory environment in primary mitral regurgitation.

BACKGROUND: Primary mitral regurgitation (PMR) is associated with oxidative and inflammatory myocardial damage. We reported greater exosome hemoglobin (Hb) in pericardial fluid (PCF) versus plasma, suggesting a cardiac source of Hb. OBJECTIVE: Test the hypothesis that Hb is produced in the PMR heart and is associated with increased inflammation. METHODS AND RESULTS: Hb gene expression for subunits alpha (HBA) and beta (HBB) was assessed in right atria (RA), left atria (LA) and left ventricular (LV) tissue from donor hearts (n = 10) and PMR patient biopsies at surgery (n = 11). PMR patients (n = 22) had PCF and blood collected for macrophage markers, pro-inflammatory cytokines, and matrix metalloproteinases (MMPs). In-situ hybridization for HBA mRNA and immunohistochemistry for Hb-alpha (Hbα) and Hb-beta (Hbß) protein was performed on PMR tissue. RESULTS: HBA and HBB genes are significantly increased (>4-fold) in RA, LA, and LV in PMR vs. normal hearts. In PMR tissue, HBA mRNA is expressed in both LV cardiomyocytes and interstitial cells by in-situ hybridization; however, Hbα and Hbß protein is only expressed in interstitial cells by immunohistochemistry. PCF oxyHb is significantly increased over plasma along with low ratios (<1.0) of haptoglobin:oxyHb and hemopexin:heme supporting a highly oxidative environment. Macrophage chemotactic protein-1, tumor necrosis factor-α, interleukin-6, and MMPs are significantly higher in PCF vs. plasma. CONCLUSION: There is increased Hb production in the PMR heart coupled with the inflammatory state of the heart, suggests a myocardial vulnerability of further Hb delivery and/or production during cardiac surgery that could adversely affect LV functional recovery.

Mitochondrial haplotype modulates genome expression and mitochondrial structure/function in cardiomyocytes following volume overload.

Mitochondrial DNA (mtDNA) haplotype regulates mitochondrial structure/function and reactive oxygen species in aortocaval fistula (ACF) in mice. Here, we unravel the mitochondrial haplotype effects on cardiomyocyte mitochondrial ultrastructure and transcriptome response to ACF in vivo. Phenotypic responses and quantitative transmission electron microscopy (TEM) and RNA sequence at 3 days were determined after sham surgery or ACF in vivo in cardiomyocytes from wild-type (WT) C57BL/6J (C57n:C57mt) and C3H/HeN (C3Hn:C3Hmt) and mitochondrial nuclear exchange mice (C57n:C3Hmt or C3Hn:C57mt). Quantitative TEM of cardiomyocyte mitochondria C3HWT hearts have more electron-dense compact mitochondrial cristae compared with C57WT. In response to ACF, mitochondrial area and cristae integrity are normal in C3HWT; however, there is mitochondrial swelling, cristae lysis, and disorganization in both C57WT and MNX hearts. Tissue analysis shows that C3HWT hearts have increased autophagy, antioxidant, and glucose fatty acid oxidation-related genes compared with C57WT. Comparative transcriptomic analysis of cardiomyocytes from ACF was dependent upon mtDNA haplotype. C57mtDNA haplotype was associated with increased inflammatory/protein synthesis pathways and downregulation of bioenergetic pathways, whereas C3HmtDNA showed upregulation of autophagy genes. In conclusion, ACF in vivo shows a protective response of C3Hmt haplotype that is in large part driven by mitochondrial nuclear genome interaction.NEW & NOTEWORTHY The results of this study support the effects of mtDNA haplotype on nuclear gene expression in cardiomyocytes. Currently, there is no acceptable therapy for volume overload due to mitral regurgitation. The findings of this study could suggest that mtDNA haplotype activates different pathways after ACF warrants further investigations on human population of heart disease from different ancestry backgrounds.

O-GlcNAcylation, novel post-translational modification linking myocardial metabolism and cardiomyocyte circadian clock.

The cardiomyocyte circadian clock directly regulates multiple myocardial functions in a time-of-day-dependent manner, including gene expression, metabolism, contractility, and ischemic tolerance. These same biological processes are also directly influenced by modification of proteins by monosaccharides of O-linked ß-N-acetylglucosamine (O-GlcNAc). Because the circadian clock and protein O-GlcNAcylation have common regulatory roles in the heart, we hypothesized that a relationship exists between the two. We report that total cardiac protein O-GlcNAc levels exhibit a diurnal variation in mouse hearts, peaking during the active/awake phase. Genetic ablation of the circadian clock specifically in cardiomyocytes in vivo abolishes diurnal variations in cardiac O-GlcNAc levels. These time-of-day-dependent variations appear to be mediated by clock-dependent regulation of O-GlcNAc transferase and O-GlcNAcase protein levels, glucose metabolism/uptake, and glutamine synthesis in an NAD-independent manner. We also identify the clock component Bmal1 as an O-GlcNAc-modified protein. Increasing protein O-GlcNAcylation (through pharmacological inhibition of O-GlcNAcase) results in diminished Per2 protein levels, time-of-day-dependent induction of bmal1 gene expression, and phase advances in the suprachiasmatic nucleus clock. Collectively, these data suggest that the cardiomyocyte circadian clock increases protein O-GlcNAcylation in the heart during the active/awake phase through coordinated regulation of the hexosamine biosynthetic pathway and that protein O-GlcNAcylation in turn influences the timing of the circadian clock.

Evidence suggesting that the cardiomyocyte circadian clock modulates responsiveness of the heart to hypertrophic stimuli in mice.

Circadian dyssynchrony of an organism (at the whole-body level) with its environment, either through light-dark (LD) cycle or genetic manipulation of clock genes, augments various cardiometabolic diseases. The cardiomyocyte circadian clock has recently been shown to influence multiple myocardial processes, ranging from transcriptional regulation and energy metabolism to contractile function. The authors, therefore, reasoned that chronic dyssychrony of the cardiomyocyte circadian clock with its environment would precipitate myocardial maladaptation to a circadian challenge (simulated shiftwork; SSW). To test this hypothesis, 2- and 20-month-old wild-type and CCM (Cardiomyocyte Clock Mutant; a model with genetic temporal suspension of the cardiomyocyte circadian clock at the active-to-sleep phase transition) mice were subjected to chronic (16-wks) biweekly 12-h phase shifts in the LD cycle (i.e., SSW). Assessment of adaptation/maladaptation at whole-body homeostatic, gravimetric, humoral, histological, transcriptional, and cardiac contractile function levels revealed essentially identical responses between wild-type and CCM littermates. However, CCM hearts exhibited increased biventricular weight, cardiomyocyte size, and molecular markers of hypertrophy (anf, mcip1), independent of aging and/or SSW. Similarly, a second genetic model of selective temporal suspension of the cardiomyocyte circadian clock (Cardiomyocyte-specific BMAL1 Knockout [CBK] mice) exhibits increased biventricular weight and mcip1 expression. Wild-type mice exhibit 5-fold greater cardiac hypertrophic growth (and 6-fold greater anf mRNA induction) when challenged with the hypertrophic agonist isoproterenol at the active-to-sleep phase transition, relative to isoproterenol administration at the sleep-to-active phase transition. This diurnal variation was absent in CCM mice. Collectively, these data suggest that the cardiomyocyte circadian clock likely influences responsiveness of the heart to hypertrophic stimuli.

Upstream stimulatory factor-2 mediates quercetin-induced suppression of PAI-1 gene expression in human endothelial cells.

The polyphenol quercetin (Quer) represses expression of the cardiovascular disease risk factor plasminogen activator inhibitor-1 (PAI-1) in cultured endothelial cells (ECs). Transfection of PAI-1 promoter-luciferase reporter deletion constructs identified a 251-bp fragment (nucleotides -800 to -549) responsive to Quer. Two E-box motifs (CACGTG), at map positions -691 (E-box1) and -575 (E-box2), are platforms for occupancy by several members of the c-MYC family of basic helix-loop-helix leucine zipper (bHLH-LZ) proteins. Promoter truncation and electrophoretic mobility shift/supershift analyses identified upstream stimulatory factor (USF)-1 and USF-2 as E-box1/E-box2 binding factors. ECs co-transfected with a 251 bp PAI-1 promoter fragment containing the two E-box motifs (p251/luc) and a USF-2 expression vector (pUSF-2/pcDNA) exhibited reduced luciferase activity versus p251/luc alone. Overexpression of USF-2 decreased, while transfection of a dominant-negative USF construct increased, EC growth consistent with the known anti-proliferative properties of USF proteins. Quer-induced decreases in PAI-1 expression and reduced cell proliferation may contribute, at least in part, to the cardioprotective benefit associated with daily intake of polyphenols.