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Bcl-2 family proteins reorganize mitochondrial membranes during apoptosis, to form pores and rearrange cristae. In vitro and in vivo analysis integrated with human genetics reveals a novel homeostatic mitochondrial function for Bcl-2 family protein Bid. Loss of full-length Bid results in apoptosis-independent, irregular cristae with decreased respiration. Bid-/- mice display stress-induced myocardial dysfunction and damage. A gene-based approach applied to a biobank, validated in two independent GWAS studies, reveals that decreased genetically determined BID expression associates with myocardial infarction (MI) susceptibility. Patients in the bottom 5% of the expression distribution exhibit >4 fold increased MI risk. Carrier status with nonsynonymous variation in Bids membrane binding domain, BidM148T, associates with MI predisposition. Furthermore, Bid but not BidM148T associates with Mcl-1Matrix, previously implicated in cristae stability; decreased MCL-1 expression associates with MI. Our results identify a role for Bid in homeostatic mitochondrial cristae reorganization, that we link to human cardiac disease.

Rapid activation causes remodeling of atrial myocytes resembling that which occurs in experimental and human atrial fibrillation (AF). Using this cellular model, we previously observed transcriptional upregulation of proteins implicated in protein misfolding and amyloidosis. For organ-specific amyloidoses such as Alzheimer's disease, preamyloid oligomers (PAOs) are now recognized to be the primary cytotoxic species. In the setting of oxidative stress, highly-reactive lipid-derived mediators known as γ-ketoaldehydes (γ-KAs) have been identified that rapidly adduct proteins and cause PAO formation for amyloid β1-42 implicated in Alzheimer's. We hypothesized that rapid activation of atrial cells triggers oxidative stress with lipid peroxidation and formation of γ-KAs, which then rapidly crosslink proteins to generate PAOs. To investigate this hypothesis, rapidly-paced and control, spontaneously-beating atrial HL-1 cells were probed with a conformation-specific antibody recognizing PAOs. Rapid stimulation of atrial cells caused the generation of cytosolic PAOs along with a myocyte stress response (e.g., transcriptional upregulation of Nppa and Hspa1a), both of which were absent in control, unpaced cells. Rapid activation also caused the formation of superoxide and γ-KA adducts in atriomyocytes, while direct exposure of cells to γ-KAs resulted in PAO production. Increased cytosolic atrial natriuretic peptide (ANP), and the generation of ANP oligomers with exposure to γ-KAs and rapid atrial HL-1 cell stimulation, strongly suggest a role for ANP in PAO formation. Salicylamine (SA) is a small molecule scavenger of γ-KAs that can protect proteins from modification by these reactive compounds. PAO formation and transcriptional remodeling were inhibited when cells were stimulated in the presence of SA, but not with the antioxidant curcumin, which is incapable of scavenging γ-KAs. These results demonstrate that γ-KAs promote protein misfolding and PAO formation as a component of the atrial cell stress response to rapid activation, and they provide a potential mechanistic link between oxidative stress and atrial cell injury.

Cytochrome (cyt) c can uncouple from the respiratory chain following mitochondrial stress and catalyze lipid peroxidation. Accumulating evidence shows that this phenomenon impairs mitochondrial respiratory function and also initiates the apoptotic cascade. Therefore, under certain conditions a pharmacological approach that can inhibit cyt c catalyzed lipid peroxidation may be beneficial. We recently showed that acetaminophen (ApAP) at normal pharmacologic concentrations can prevent hemoprotein-catalyzed lipid peroxidation in vitro and in vivo by reducing ferryl heme to its ferric state. We report here, for the first time, that ApAP inhibits cytochrome c-catalyzed oxidation of unsaturated free fatty acids and also the mitochondrial phospholipid, cardiolipin. Using isolated mitochondria, we also showed that ApAP inhibits cardiolipin oxidation induced by the pro-apoptotic protein, tBid. We found that the IC(50) of the inhibition of cardiolipin oxidation by ApAP is similar in both intact isolated mitochondria and cardiolipin liposomes, suggesting that ApAP penetrates well into the mitochondria. Together with our previous results, the findings presented herein suggest that ApAP is a pleiotropic inhibitor of peroxidase catalyzed lipid peroxidation. Our study also provides a potentially novel pharmacological approach for inhibiting the cascade of events that can result from redox cycling of cyt c.

Platelet activation is important in the regulation of hemostasis and thrombosis. Uncontrolled activation of platelets may lead to arterial thrombosis, which is a major cause of myocardial infarction and stroke. After activation, metabolism of arachidonic acid (AA) by 12-lipoxygenase (12-LOX) may play a significant role in regulating the degree and stability of platelet activation because inhibition of 12-LOX significantly attenuates platelet aggregation in response to various agonists. Protein kinase C (PKC) activation is also known to be an important regulator of platelet activity. Using a newly developed selective inhibitor for 12-LOX and a pan-PKC inhibitor, we investigated the role of PKC in 12-LOX-mediated regulation of agonist signaling in the platelet. To determine the role of PKC within the 12-LOX pathway, a number of biochemical endpoints were measured, including platelet aggregation, calcium mobilization, and integrin activation. Inhibition of 12-LOX or PKC resulted in inhibition of dense granule secretion and attenuation of both aggregation and αIIbβ(3) activation. However, activation of PKC downstream of 12-LOX inhibition rescued agonist-induced aggregation and integrin activation. Furthermore, inhibition of 12-LOX had no effect on PKC-mediated aggregation, indicating that 12-LOX is upstream of PKC. These studies support an essential role for PKC downstream of 12-LOX activation in human platelets and suggest 12-LOX as a possible target for antiplatelet therapy.

Cytosolic phospholipase [A.sub.2[alpha]] ([cPLA.sub.2[alpha]]) hydrolyzes arachidonic acid from cellular membrane phospholipids, thereby providing enzymatic substrates for the synthesis of eicosanoids, such as prostaglandins and leukotrienes. Considerable understanding of [cPLA.sub.2[alpha]] function has been derived from investigations of the enzyme and from [cPLA.sub.2[alpha]]-null mice, but knowledge of discrete roles for this enzyme in humans is limited. We investigated a patient hypothesized to have an inherited prostanoid biosynthesis deficiency due to his multiple, complicated small intestinal ulcers despite no use of cyclooxygenase inhibitors. Levels of thromboxane [B.sub.2] and 12-hydroxyeicosatetraenoic acid produced by platelets and leukotriene [B.sub.4] released from calcium ionophore--activated blood were markedly reduced, indicating defective enzymatic release of the arachidonic acid substrate for the corresponding cyclooxygenase and lipoxygenases. Platelet aggregation and degranulation induced by adenosine diphosphate or collagen were diminished but were normal in response to arachidonic acid. Two heterozygous single base pair mutations and a known SNP were found in the coding regions of the patient's [cPLA.sub.2[alpha]] genes (p.[Ser111Pro]+[Arg485His; Lys651Arg]). The total [PLA.sub.2] activity in sonicated platelets was diminished, and the urinary metabolites of prostacyclin, prostaglandin [E.sub.2], prostaglandin [D.sub.2], and thromboxane [A.sub.2] were also reduced. These findings characterize what we believe is a novel inherited deficiency of [cPLA.sub.2]., Introduction These investigations addressed the problem of a patient with multiple small intestinal ulcers that were not associated with use of NSAIDs. The patient had chronic gastrointestinal blood loss from [...]