Alcohol in moderation, cardioprotection, and neuroprotection: epidemiological considerations and mechanistic studies.

In contrast to many years of important research and clinical attention to the pathological effects of alcohol (ethanol) abuse, the past several decades have seen the publication of a number of peer-reviewed studies indicating the beneficial effects of light-moderate, nonbinge consumption of varied alcoholic beverages, as well as experimental demonstrations that moderate alcohol exposure can initiate typically cytoprotective mechanisms. A considerable body of epidemiology associates moderate alcohol consumption with significantly reduced risks of coronary heart disease and, albeit currently a less robust relationship, cerebrovascular (ischemic) stroke. Experimental studies with experimental rodent models and cultures (cardiac myocytes, endothelial cells) indicate that moderate alcohol exposure can promote anti-inflammatory processes involving adenosine receptors, protein kinase C (PKC), nitric oxide synthase, heat shock proteins, and others which could underlie cardioprotection. Also, brain functional comparisons between older moderate alcohol consumers and nondrinkers have received more recent epidemiological study. In over half of nearly 45 reports since the early 1990s, significantly reduced risks of cognitive loss or dementia in moderate, nonbinge consumers of alcohol (wine, beer, liquor) have been observed, whereas increased risk has been seen only in a few studies. Physiological explanations for the apparent CNS benefits of moderate consumption have invoked alcohol's cardiovascular and/or hematological effects, but there is also experimental evidence that moderate alcohol levels can exert direct "neuroprotective" actions-pertinent are several studies in vivo and rat brain organotypic cultures, in which antecedent or preconditioning exposure to moderate alcohol neuroprotects against ischemia, endotoxin, beta-amyloid, a toxic protein intimately associated with Alzheimer's, or gp120, the neuroinflammatory HIV-1 envelope protein. The alcohol-dependent neuroprotected state appears linked to activation of signal transduction processes potentially involving reactive oxygen species, several key protein kinases, and increased heat shock proteins. Thus to a certain extent, moderate alcohol exposure appears to trigger analogous mild stress-associated, anti-inflammatory mechanisms in the heart, vasculature, and brain that tend to promote cellular survival pathways.

Signals from type 1 sphingosine 1-phosphate receptors enhance adult mouse cardiac myocyte survival during hypoxia.

Sphingosine 1-phosphate (S1P) is a biologically active lysophospholipid that serves as a key regulator of cellular differentiation and survival. Immune stimuli increase S1P synthesis and secretion by mast cells and platelets, implicating this molecule in tissue responses to injury and inflammation. Binding of S1P to G(i) protein-coupled receptors activates phosphatidylinositol 3-kinase and Akt in a variety of tissues. To elucidate the mechanisms by which S1P enhances adult cardiac myocyte survival during hypoxia, we used a mouse cell culture system in which S1P(1) receptors were observed to transduce signals from exogenous S1P, an S1P(1) receptor antibody with agonist properties, and the pharmacological agents FTY720 and SEW2871. S1P(1) receptor mRNA and protein were abundantly expressed by adult mouse cardiac myocytes. S1P-S1P(1) receptor axis enhancement of myocyte survival during hypoxia was abolished by phosphatidylinositol 3-kinase inhibition. S1P(1) receptor function was closely associated with activation of Akt, inactivation of GSK-3beta, and reduction of cytochrome c release from heart mitochondria. These observations highlight the importance of S1P(1) receptors on ventricular myocytes as mediators of inducible resistance against cellular injury during severe hypoxic stress.

Transgenic MMP-2 expression induces latent cardiac mitochondrial dysfunction.

Matrix metalloproteinases (MMPs) are central to the development and progression of dysfunctional ventricular remodeling after tissue injury. We studied 6 month old heterozygous mice with cardiac-specific transgenic expression of active MMP-2 (MMP-2 Tg). MMP-2 Tg hearts showed no substantial gross alteration of cardiac phenotype compared to age-matched wild-type littermates. However, buffer perfused MMP-2 Tg hearts subjected to 30 min of global ischemia followed by 30 min of reperfusion had a larger infarct size and greater depression in contractile performance compared to wild-type hearts. Importantly, cardioprotection mediated by ischemic preconditioning (IPC) was completely abolished in MMP-2 Tg hearts, as shown by abnormalities in mitochondrial ultrastructure and impaired respiration, increased lipid peroxidation, cell necrosis and persistently reduced recovery of contractile performance during post-ischemic reperfusion. We conclude that MMP-2 functions not only as a proteolytic enzyme but also as a previously unrecognized active negative regulator of mitochondrial function during superimposed oxidative stress.

Poly(ADP-ribose) polymerase-1 hyperactivation and impairment of mitochondrial respiratory chain complex I function in reperfused mouse hearts.

Poly(ADP-ribose) polymerase-1 (PARP-1), the most abundant member of the PARP family, is a nuclear enzyme that catalyzes ADP-ribose transfer from NAD+ to specific acceptor proteins in response to DNA damage. Excessive PARP-1 activation is an important cause of infarction and contractile dysfunction in heart tissue during interruptions of blood flow. The mechanisms by which PARP-1 inhibition and disruption dramatically improve metabolic recovery and reduce oxidative stress during cardiac reperfusion have not been fully explored. We developed a mouse heart experimental protocol to test the hypothesis that mitochondrial respiratory complex I is a downstream mediator of beneficial effects of PARP-1 inhibition or disruption. Pharmacological inhibition of PARP-1 activity produced no deterioration of hemodynamic function in C57BL/6 mouse hearts. Hearts from PARP-1 knockout mice also exhibited normal baseline contractility. Prolonged ischemia-reperfusion produced a selective defect in complex I function distal to the NADH dehydrogenase component. PARP-1 inhibition and PARP-1 gene disruption conferred equivalent protection against mitochondrial complex I injury and were strongly associated with improvement in myocardial energetics, contractility, and tissue viability. Interestingly, ischemic preconditioning abolished cardioprotection stimulated by PARP-1 gene disruption. Treatment with the antioxidant N-(2-mercaptopropionyl)-glycine or xanthine oxidase inhibitor allopurinol restored the function of preconditioned PARP-1 knockout hearts. This investigation establishes a strong association between PARP-1 hyperactivity and mitochondrial complex I dysfunction in cardiac myocytes. Our findings advance understanding of metabolic regulation in myocardium and identify potential therapeutic targets for prevention and treatment of ischemic heart disease.

MnSOD in mouse heart: acute responses to ischemic preconditioning and ischemia-reperfusion injury.

Manganese superoxide dismutase (MnSOD) is one of the main antioxidant enzymes that protects the heart against ischemia-reperfusion (I/R) injury. Ischemic preconditioning (IPC) is a short period of ischemia-reperfusion that reduces subsequent prolonged I/R injury. Although MnSOD localizes in mitochondria, the immediate subcellular distribution of MnSOD in heart after IPC and I/R has not been studied. In a Langendorff mouse heart model, IPC significantly improved cardiac function and reduced the infarction size induced by I/R. Immunoblotting and double immunostaining in fresh preparations revealed that I/R resulted in an increase in cytosolic MnSOD content accompanied by the release of cytochrome c. In contrast, IPC increased mitochondrial MnSOD and reduced cytosolic MnSOD and cytochrome c release induced by I/R. We found that compared with freshly prepared fractions, the freeze-thaw approach results in mitochondrial integrity disruption and release of large amounts of MnSOD into the cytosol along with mitochondrial markers even in the absence of I/R. In contrast, fresh preparations exhibit early MnSOD release into the cytosol after I/R that is prevented by IPC and cyclosporin A administration.

Preservation of base-line hemodynamic function and loss of inducible cardioprotection in adult mice lacking protein kinase C epsilon.

Signaling pathways involving protein kinase C isozymes are modulators of cardiovascular development and response to injury. Protein kinase C epsilon activation in cardiac myocytes reduces necrosis caused by coronary artery disease. However, it is unclear whether protein kinase C epsilon function is required for normal cardiac development or inducible protection against oxidative stress. Protein kinase C delta activation is also observed during cardiac preconditioning. However, its role as a promoter or inhibitor of injury is controversial. We examined hearts from protein kinase C epsilon knock-out mice under physiological conditions and during acute ischemia reperfusion. Null-mutant and wild-type mice displayed equivalent base-line morphology and hemodynamic function. Targeted disruption of the protein kinase C epsilon gene blocked cardioprotection caused by ischemic preconditioning and alpha(1)-adrenergic receptor stimulation. Protein kinase C delta activation increased in protein kinase C epsilon knock-out myocytes without altering resistance to injury. These observations support protein kinase C epsilon activation as an essential component of cardioprotective signaling. Our results favor protein kinase C delta activation as a mediator of normal growth. This study advances the understanding of cellular mechanisms responsible for preservation of myocardial integrity as potential targets for prevention and treatment of ischemic heart disease.

Effect of anoxia/reperfusion on the reversible active/de-active transition of NADH-ubiquinone oxidoreductase (complex I) in rat heart.

The multi-subunit mammalian NADH-ubiquinone oxidoreductase (complex I) is part of the mitochondrial electron transport chain and physiologically serves to reduce ubiquinone with NADH as the electron donor. The three-dimensional structure of this enzyme complex remains to be elucidated and also little is known about the physiological regulation of complex I. The enzyme complex in vitro is known to exist as a mixture of active (A) and de-active (D) forms [Biochim. Biophys. Acta 1364 (1998) 169]. Studies are reported here examining the effect of anoxia and reperfusion on the A/D-equilibrium of complex I in rat hearts ex vivo. Complex I from the freshly isolated rat heart or after prolonged (1 h) normoxic perfusion exists in almost fully active form (87+/-2%). Either 30 min of nitrogen perfusion or global ischemia decreases the portion of active form of complex I to 40+/-2%. Upon re-oxygenation of cardiac tissue, complex I is converted back predominantly to the active form (80-85%). Abrupt alternation of anoxic and normoxic perfusion allows cycling between the two states of the enzyme. The possible role in the physiological regulation of complex I activity is discussed.

Moderate alcohol consumption induces sustained cardiac protection by activating PKC-epsilon and Akt.

C57BL/6 mice were fed 18% ethanol (vol/vol) in drinking water for 12 wk. Isovolumic hearts were subjected to 20 min of ischemia and 30 min of reperfusion on a Langendorff apparatus. There were no differences in baseline hemodynamic function between hearts from ethanol (EtOH)-fed mice and controls. However, prior alcohol consumption doubled recovery of left ventricular developed pressure (68 +/- 8 vs. 33 +/- 8 mmHg for controls; n = 10, P < 0.05) and reduced creatine kinase release by half (0.26 +/- 0.04 vs. 0.51 +/- 0.08 U x min(-1) x g wet wt(-1) for controls; n = 10, P < 0.05). EtOH feeding doubled expression of activated protein kinase C epsilon (PKC)epsilon (n = 6, P < 0.05); whereas PKC inhibition blocked protection during ischemia-reperfusion. EtOH feeding also increased expression of Akt three- to fivefold (n = 6, P < 0.05), whereas PKC inhibition prevented increases in Akt kinase activity. We conclude that signaling pathways involving PKC-epsilon are critical for sustained EtOH-mediated cardioprotection and that Akt may be a downstream effector of resistance to myocardial reperfusion injury.

Cardioprotection mediated by sphingosine-1-phosphate and ganglioside GM-1 in wild-type and PKC epsilon knockout mouse hearts.

Sphingosine-1-phosphate (S1P) protects neonatal rat cardiac myocytes from hypoxic damage through unknown signaling pathways. We tested the hypothesis that S1P-induced cardioprotection requires activation by the epsilon-isoform of protein kinase C (PKC epsilon) by subjecting hearts isolated from PKC epsilon knockout mice and wild-type mice to 20 min of global ischemia and 30 min of reperfusion. Pretreatment with a 2-min infusion of 10 nM S1P improved recovery of left ventricular developed pressure (LVDP) in both wild-type and PKC epsilon knockout hearts and reduced the rise in LV end-diastolic pressure (LVEDP) and creatine kinase (CK) release. Pretreatment for 2 min with 10 nM of the ganglioside GM-1 also improved recovery of LVDP and suppressed CK release in wild-type hearts but not in PKC epsilon knockout hearts. Importantly, GM-1 but not S1P, increased the proportion of PKC epsilon localized to particulate fractions. Our results suggest that GM-1, which enhances endogenous S1P production, reduces cardiac injury through PKC epsilon-dependent intracellular pathways. In contrast, extracellular S1P induces equivalent cardioprotection through PKC epsilon-independent signaling pathways.