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Several interventions, such as ischemic preconditioning, remote pre/perconditioning, or postconditioning, are known to decrease lethal myocardial ischemia-reperfusion injury. While several signal transduction pathways become activated by such maneuvers, they all have a common end point, namely, the mitochondria. These organelles represent an essential target of the cardioprotective strategies, and the preservation of mitochondrial function is central for the reduction of ischemia-reperfusion injury. In the present review, we address the role of mitochondria in the different conditioning strategies; in particular, we focus on alterations of mitochondrial function in terms of energy production, formation of reactive oxygen species, opening of the mitochondrial permeability transition pore, and mitochondrial dynamics induced by ischemia-reperfusion.

Although incidence and prevalence of prediabetes are increasing, little is known about its cardiac effects. Therefore, our aim was to investigate the effect of prediabetes on cardiac function and to characterize parameters and pathways associated with deteriorated cardiac performance. Long-Evans rats were fed with either control or high-fat chow for 21 wk and treated with a single low dose (20 mg/kg) of streptozotocin at week 4 High-fat and streptozotocin treatment induced prediabetes as characterized by slightly elevated fasting blood glucose, impaired glucose and insulin tolerance, increased visceral adipose tissue and plasma leptin levels, as well as sensory neuropathy. In prediabetic animals, a mild diastolic dysfunction was observed, the number of myocardial lipid droplets increased, and left ventricular mass and wall thickness were elevated; however, no molecular sign of fibrosis or cardiac hypertrophy was shown. In prediabetes, production of reactive oxygen species was elevated in subsarcolemmal mitochondria. Expression of mitofusin-2 was increased, while the phosphorylation of phospholamban and expression of Bcl-2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP3, a marker of mitophagy) decreased. However, expression of other markers of cardiac auto- and mitophagy, mitochondrial dynamics, inflammation, heat shock proteins, Ca 2+ /calmodulin-dependent protein kinase II, mammalian target of rapamycin, or apoptotic pathways were unchanged in prediabetes. This is the first comprehensive analysis of cardiac effects of prediabetes indicating that mild diastolic dysfunction and cardiac hypertrophy are multifactorial phenomena that are associated with early changes in mitophagy, cardiac lipid accumulation, and elevated oxidative stress and that prediabetes-induced oxidative stress originates from the subsarcolemmal mitochondria., (Copyright © 2016 the American Physiological Society.)

Cardiomyocyte dedifferentiation may be an important source of proliferating cardiomyocytes facilitating cardiac repair. Cardiomyocyte dedifferentiation and proliferation induced by oncostatin-M (OSM) is characterized by sarcomere degeneration. However, the mechanism underlying sarcomere degeneration remains unclear. We hypothesized that this process may involve matrix metalloproteinase-2 (MMP-2), a key protease localized at the sarcomere in cardiomyocytes. We tested the hypothesis that MMP-2 is involved in the sarcomere degeneration that characterizes cardiomyocyte dedifferentiation. Confocal immunofluorescence and biochemical methods were used to explore the role of MMP-2 in OSM-induced dedifferentiation of neonatal rat ventricular myocytes (NRVM). OSM caused a concentration- and time-dependent loss of sarcomeric α-actinin and troponin-I in NRVM. Upon OSM-treatment, the mature sarcomere transformed to a phenotype resembling a less-developed sarcomere, i.e., loss of sarcomeric proteins and Z-disk transformed into disconnected Z bodies, characteristic of immature myofibrils. OSM dose dependently increased MMP-2 activity. Both the pan-MMP inhibitor GM6001 and the selective MMP-2 inhibitor ARP 100 prevented sarcomere degeneration induced by OSM treatment. OSM also induced NRVM cell cycling and increased methyl-thiazolyl-tetrazolium (MTT) staining, preventable by MMP inhibition. These results suggest that MMP-2 mediates sarcomere degeneration in OSM-induced cardiomyocyte dedifferentiation and thus potentially contributes to cardiomyocyte regeneration., (Copyright © 2016 the American Physiological Society.)

Matrix metalloproteinase-2 (MMP-2) has been extensively studied in the context of extracellular matrix remodeling but is also localized within cells and can be activated by prooxidants to proteolyze specific intercellular targets. Although there are reports of MMP-2 in mitochondria, a critical source of cellular oxidative stress, these studies did not take into account the presence within their preparations of the mitochondria-associated membrane (MAM), a subdomain of the endoplasmic reticulum (ER). We hypothesized that MMP-2 is situated in the MAM and therefore investigated its subcellular distribution between mitochondria and the MAM. Immunogold electron microscopy revealed MMP-2 localized in mitochondria of heart sections from mice. In contrast, immunofluorescence analysis of an MMP-2:HaloTag fusion protein expressed in HL-1 cardiomyocytes showed an ER-like distribution, with greater colocalization with an ER marker (protein disulfide isomerase) relative to the mitochondrial marker, MitoTracker red. Although MMP-2 protein and enzymatic activity were present in crude mitochondrial fractions, once these were separated into purified mitochondria and MAM, MMP-2 was principally associated with the latter. Thus, although mitochondria may contain minimal levels of MMP-2, the majority of MMP-2 previously identified as "mitochondrial" is in fact associated with the MAM. We also found that calreticulin, an ER- and MAM-resident Ca(2+) handling protein and chaperone, could be proteolyzed by MMP-2 in vitro. MAM-localized MMP-2 could therefore potentially impact mitochondrial function by affecting ER-mitochondrial Ca(2+) signaling via its proteolysis of calreticulin.

Cardioprotection by ischemic preconditioning (IP) was abolished in connexin 43 (Cx43)-deficient mice due to loss of Cx43 located in mitochondria rather than at the sarcolemma. IP is lost in hyperlipidemic rat hearts as well. Since changes in mitochondrial Cx43 in hyperlipidemia have not yet been analyzed, we determined total and mitochondrial Cx43 levels in male Wistar rats fed a laboratory chow enriched with 2% cholesterol or normal chow for 12 wk. Hearts were isolated and perfused according to Langendorff. After a 10-min perfusion, myocardial tissue cholesterol, superoxide, and nitrotyrosine contents were measured and Cx43 content in whole heart homogenate and a mitochondrial fraction determined. In the cholesterol-fed group, tissue cholesterol and superoxide formation was increased (P < 0.05), while total Cx43 content remained unchanged. Mitochondrial total and dephosphorylated Cx43 content decreased. Hearts were subjected to an IP protocol (3 × 5 min ischemia-reperfusion) or time-matched aerobic perfusion followed by 30-min global ischemia and 5-min reperfusion. IP reduced infarct size in normal but not in cholesterol-fed rats. At 5-min reperfusion following 30-min global ischemia, the total and dephosphorylated mitochondrial Cx43 content was increased, which was abolished by IP in both normal and high-cholesterol diet. In conclusion, loss of cardioprotection by IP in hyperlipidemia is associated with a redistribution of both sarcolemmal and mitochondrial Cx43.

Matrix metalloproteinases (MMPs) are known to degrade components of the extracellular matrix. More recently, in myocardial oxidative stress injury including ischemia-reperfusion, MMP-2 is activated and degrades troponin I and α-actinin. MMP activity is regulated at several levels. We recently showed that MMP-2 is localized in the caveolae of cardiomyocytes and is negatively regulated by caveolin-1 (Cav-1). The caveolin scaffolding domain of Cav-1 inhibits MMP-2 proteolytic activity in vitro, and Cav-1(-/-) mouse hearts have increased MMP-2 activity compared with controls. Whether this increase in MMP-2 activity translates to impaired cardiac function is unknown. Hearts isolated from Cav-1(-/-) mice and their wild-type controls were perfused as isolated working hearts and physiologically challenged with increasing increments of left atrial preload (7-22.5 mmHg). The hearts were then pharmacologically challenged with increasing concentrations of isoproterenol (0.1-100 nM). Functionally, the Cav-1(-/-) hearts were similar to the controls in heart rate, peak systolic pressure, developed pressure, and rate pressure product. At higher preload pressures, the Cav-1(-/-) hearts outperformed the control hearts. Coronary flow was significantly higher in Cav-1(-/-) hearts under all conditions. The highest concentration of isoproternol increased the heart rate of Cav-1(-/-) hearts more than in controls. Western blot analysis revealed no significant changes in troponin I or α-actinin between Cav-1(-/-) hearts and their controls. There was a significant loss of MMP-2 from both knockout and control hearts during the perfusion. In summary, despite the loss of Cav-1, Cav-1(-/-) hearts show similar or better cardiac function compared with wild-type hearts following physiological challenge or β-adrenergic stimulation in vitro, and this appears unrelated to changes in MMP-2.

Our objective was to address the balance of inducible nitric oxide (NO) synthase (iNOS) and arginase and their contribution to contractile dysfunction in heart failure (HF). Excessive NO formation is thought to contribute to contractile dysfunction; in macrophages, increased iNOS expression is associated with increased arginase expression, which competes with iNOS for arginine. With substrate limitation, iNOS may become uncoupled and produce reactive oxygen species (ROS). In rabbits, HF was induced by left ventricular (LV) pacing (400 beats/min) for 3 wk. iNOS mRNA [quantitative real-time PCR (qRT-PCR)] and protein expression (confocal microscopy) were detected, and arginase II expression was quantified with Western blot; serum arginine and myocardial nitrite and nitrate concentrations were determined by chemiluminescence, and protein S-nitrosylation with Western blot. Superoxide anions were quantified with dihydroethidine staining. HF rabbits had increased LV end-diastolic diameter [20.0 + or - 0.5 (SE) vs. 17.2 + or - 0.3 mm in sham] and decreased systolic fractional shortening (11.1 + or - 1.4 vs. 30.6 + or - 0.7% in sham; both P < 0.05). Myocardial iNOS mRNA and protein expression were increased, however, not associated with increased myocardial nitrite or nitrate concentrations or protein S-nitrosylation. The serum arginine concentration was decreased (124.3 + or - 5.6 vs. 155.4 + or - 12.0 micromol/l in sham; P < 0.05) at a time when cardiac arginase II expression was increased (0.06 + or - 0.01 vs. 0.02 + or - 0.01 arbitrary units in sham; P < 0.05). Inhibition of iNOS with 1400W attenuated superoxide anion formation and contractile dysfunction in failing hearts. Concomitant increases in iNOS and arginase expression result in unchanged NO species and protein S-nitrosylation; with substrate limitation, uncoupled iNOS produces superoxide anions and contributes to contractile dysfunction.

Persistent arterial hypotension is a hallmark of sepsis and is believed to be caused, at least in part, by excess nitric oxide (NO). NO can combine with superoxide to produce peroxynitrite, which activates matrix metalloproteinases (MMPs). Whether MMP inhibition in vivo protects against vascular hyporeactivity induced by endotoxemia is unknown. Male Sprague-Dawley rats were administered either bacterial lipopolysaccharide (LPS, 4 mg/kg ip) or vehicle (pyrogen-free water). Later (30 min), animals received the MMP inhibitor doxycycline (4 mg/kg ip) or vehicle (pyrogen-free water). After LPS injection (6 h), animals were killed, and aortas were excised. Aortic rings were mounted in organ baths, and contractile responses to phenylephrine or KCl were measured. Aortas and plasma were examined for MMP activity by gelatin zymography. Aortic MMP and inducible nitric oxide synthase (iNOS) were examined by immunoblot and/or immunohistochemistry. Doxycycline prevented the LPS-induced development of ex vivo vascular hyporeactivity to phenylephrine and KCl. iNOS protein was significantly upregulated in aortic homogenates from endotoxemic rats; doxycycline did not alter its level. MMP-9 activity was undetectable in aortic homogenates from LPS-treated rats but significantly upregulated in the plasma; this was attenuated by doxycycline. Plasma MMP-2 activities were unchanged by LPS. Specific MMP-2 activity was increased in aortas from LPS-treated rats. This study demonstrates the in vivo protective effect of the MMP inhibitor doxycycline against the development of vascular hyporeactivity in endotoxemic rats.

Cardiac connexin 43 (Cx43) is involved in infarct propagation, and the uncoupling of Cx43-formed channels reduces infarct size. Cx43-formed channels open upon Cx43 dephosphorylation, and ischemic preconditioning (IP) prevents the ischemia-induced Cx43 dephosphorylation. In addition to the sarcolemma, Cx43 is also present in the cardiomyocyte mitochondria. We now examined the interaction of Cx43 with protein phosphatases PP1alpha, PP2Aalpha, and PP2Balpha and the role of such interaction for Cx43 phosphorylation in preconditioned myocardium. Infarct size (in %area at risk) in left ventricular anterior myocardium of Göttinger minipigs subjected to 90 min of low-flow ischemia and 120 min of reperfusion was 23.1 +/- 2.7 [n = 7, nonpreconditioned (NIP) group] and was reduced by IP to 10.0 +/- 3.2 (n = 6, P < 0.05). Mitochondrial and gap junctional Cx43 dephosphorylation increased after 85 min of ischemia in NIP myocardium, whereas Cx43 phosphorylation was preserved with IP. PP2Aalpha and PP1alpha, but not PP2Balpha, were detected by Western blot analysis in the left ventricular myocardium. Cx43 coprecipitated with PP2Aalpha but not with PP1alpha. Although the total PP2Aalpha immunoreactivity (confocal laser scan) was increased to 154 +/- 24% and 194 +/- 13% of baseline (P < 0.05) after 85 min of ischemia in NIP and IP myocardium, respectively, the PP2A activities were similar between the groups. The amount of PP2Aalpha coimmunoprecipitated with Cx43 remained unchanged. Only PP2Aalpha coprecipitates with Cx43 in pig myocardium. This interaction is not affected by IP, suggesting that PP2Aalpha is not involved in the prevention of the ischemia-induced Cx43 dephosphorylation by IP.

Multiparity is associated with increased risk of cardiovascular disease. We tested whether multiparity induces oxidative stress in rat vascular tissue. Coronary arteries and thoracic aorta were isolated from multiparous and age-matched virgin rats. Relaxation to ACh and sodium nitroprusside (SNP) was measured by wire myography. We also tested the effect of the superoxide dismutase mimetic MnTE2PyP (30 microM), the NADPH oxidase inhibitor apocynin (10 microM), and the peroxynitrite scavenger FeTPPs (10 microM) on ACh-mediated relaxation in coronary arteries. Vascular superoxide anion was measured using the luminol derivative L-012 and nitric oxide (NO) generation by the Griess reaction. Multiparity reduced maximal response and sensitivity to ACh in coronary arteries [maximal relaxation (E(max)): multiparous 49+/-3% vs. virgins 95%+/-3%; EC(50): multiparous 135+/-1 nM vs. virgins 60+/-1 nM], and in aortic rings (E(max): multiparous 38+/-3% vs. virgins 79+/-4%; EC(50): multiparous 160+/-2 nM vs. virgins 90+/-3 nM). Coronary arteries from the two groups relaxed similarly to SNP. Superoxide anions formation was significantly higher in both coronary arteries (2.8-fold increase) and aorta (4.1-fold increase) from multiparous rats compared with virgins. In multiparous rats, incubation with MnTE2PyP, apocynin, and FeTPPs improved maximal relaxation to ACh (MnTE2PyP: 74+/-5%; vehicle: 41+/-5%; apocynin: 73+/-3% vs. vehicle: 41+/-3%; FeTPPs: 72+/-3% vs. vehicle: 46+/-3%) and increased sensitivity (EC(50): MnTE2PyP: 61+/-0.5 nM vs. vehicle: 91+/-1 nM; apocynin: 45+/-3 nM vs. vehicle: 91+/-6 nM; FeTPP: 131 +/- 2 nM vs. vehicle: 185+/-1 nM). Multiparity also reduced total nitrate/nitrite levels (multiparous: 2.5+/-2 micromol/mg protein vs. virgins: 7+/-1 micromol/mg protein) and endothelial nitric oxide synthase protein levels (multiparous: 0.53+/-0.1 protein/actin vs. virgins: 1.0+/-0.14 protein/actin). These data suggest that multiparity induces endothelial dysfunction through decreased NO bioavailability and increased reactive oxygen species formation.

Connexin 43 (Cx43) is localized at left ventricular (LV) gap junctions and in cardiomyocyte mitochondria. A genetically induced reduction of Cx43 as well as blockade of mitochondrial Cx43 import abolishes the infarct size (IS) reduction by ischemic preconditioning (IP). With progressing age, Cx43 content in ventricular and atrial tissue homogenates is reduced. We now investigated whether or not 1) the mitochondrial Cx43 content is reduced in aged mice hearts and 2) IS reduction by IP is lost in aged mice hearts in vivo. Confirming previous results, sarcolemmal Cx43 content was reduced in aged (>13 mo) compared with young (<3 mo) C57Bl/6 mice hearts, whereas the expression levels of protein kinase C epsilon and endothelial nitric oxide synthase remained unchanged. Also in mitochondria isolated from aged mice LV myocardium, Western blot analysis indicated a 40% decrease in Cx43 content compared with mitochondria isolated from young mice hearts. In young mice hearts, IP by one cycle of 10 min ischemia and 10 min reperfusion reduced IS (% of area at risk) following 30 min regional ischemia and 120 min reperfusion from 67.7 +/- 3.3 (n = 17) to 34.2 +/- 6.6 (n = 5, P < 0.05). In contrast, IP's cardioprotection was lost in aged mice hearts, since IS in nonpreconditioned (57.5 +/- 4.0, n = 10) and preconditioned hearts (65.4 +/- 6.3, n = 8, P = not significant) was not different. In conclusion, mitochondrial Cx43 content is decreased in aged mouse hearts. The reduced levels of Cx43 may contribute to the age-related loss of cardioprotection by IP.

Protection from a prolyl hydroxylase domain-containing enzyme (PHD) inhibitor, desferoxamine (DFO), was recently reported to be dependent on production of reactive oxygen species (ROS). Ischemic preconditioning triggers the protected state by stimulating nitric oxide (NO) production to open mitochondrial ATP-sensitive K+ (mitoK(ATP)) channels, generating ROS required for protection. We tested whether DFO and a second PHD inhibitor, ethyl-3,4-dihydroxybenzoate (EDHB), might have similar mechanisms. EDHB and DFO increased ROS generation by 50-75% (P < 0.001) in isolated rabbit cardiomyocytes. This increase after EDHB exposure was blocked by N(omega)-nitro-L-arginine methyl ester (L-NAME), an NO synthase (NOS) inhibitor; ODQ, a guanylyl cyclase antagonist; and Rp-8-bromoguanosine-3',5'-cyclic monophosphorothioate Rp isomer, a PKG blocker, thus implicating the NO pathway in EDHB's signaling. Glibenclamide, a nonselective K(ATP) channel blocker, or 5-hydroxydecanoate, a selective mitoK(ATP) channel antagonist, also prevented EDHB's ROS production, as did blockade of mitochondrial electron transport with myxothiazol. NOS is activated by Akt. However, neither wortmannin, an inhibitor of phosphatidylinositol-3-kinase, nor Akt inhibitor blocked EDHB-induced ROS generation, indicating that EDHB initiates signaling downstream of Akt. DFO also increased ROS production, and this effect was blocked by ODQ, 5-hydroxydecanoate, and N-(2-mercaptopropionyl)glycine, an ROS scavenger. DFO increased cardiomyocyte production of nitrite, a metabolite of NO, and this effect was blocked by an inhibitor of NOS. DFO also spared ischemic myocardium in intact hearts. This infarct-sparing effect was blocked by ODQ, L-NAME, and N-(2-mercaptopropionyl)glycine. Hence, DFO and EDHB stimulate NO-dependent activation of PKG to open mitoK(ATP) channels and produce ROS, which act as second messengers to trigger entrance into the preconditioned state.

Chronic coronary artery stenosis results in patchy necrosis in the dependent myocardium and impairs global and regional left ventricular (LV) function in rats in vivo. The aim of the present study was to compare regional myocardial blood flow (RMBF) and function (F) in poststenotic myocardium by using magnetic resonance imaging (MRI) and to compare MRI blood flow changes to histological alterations to assess whether RMBF in the viable poststenotic tissue remains normal. MRI was performed in 11 anesthetized Wistar rats with 2-wk stenosis of the left coronary artery. Postmortem, the extent of fibrotic tissue was quantified. Poststenotic RMBF was significantly reduced to 2.21 +/- 0.30 ml.g(-1).min(-1) compared with RMBF in the remote myocardium (4.05 +/- 0.50 ml.g(-1).min(-1)). A significant relationship between the poststenotic RMBF (%remote area) and the poststenotic F (%remote myocardium) was calculated (r = 0.61, P < 0.05). Assuming perfusion in scar tissue to be 32 +/- 5% of perfusion of remote myocardium, as measured in five additional rats, and that in remote myocardium to be 114 +/- 25% of that in normal myocardium, as assessed in five sham rats, the calculated perfusion in partially fibrotic tissue samples (35.7 +/- 5.2% of analyzed area) was 2.88 +/- 0.18 ml.g(-1).min(-1), whereas measured MRI perfusion was only 1.86 +/- 0.24 ml.g(-1).min(-1) (P < 0.05). These results indicate that resting perfusion in viable poststenotic myocardium is moderately reduced. Alterations in global and regional LV function are therefore secondary to both patchy fibrosis and reduced resting perfusion.

The pathophysiology of myocardial hibernation is characterized as a situation of reduced regional contractile function distal to a coronary artery stenosis that recovers after removal of the coronary stenosis. A subacute "downregulation" of contractile function in response to reduced regional myocardial blood flow exists, which normalizes regional energy and substrate metabolism but does not persist for more than 12-24 h. Chronic hibernation develops in response to one or more episodes of myocardial ischemia-reperfusion, possibly progressing from repetitive stunning with normal blood flow to hibernation with reduced blood flow. An upregulation of a protective gene program is seen in hibernating myocardium, putting it into the context of preconditioning. The morphology of hibernating myocardium is characterized by both adaptive and degenerative features.

Chronic hypoxia may precondition the myocardium and protect from ischemia-reperfusion damage. We therefore examined the recovery of left and right ventricular function after ischemia and reperfusion (15 min each) in isolated blood-perfused working hearts from normoxic (Norm) and hypoxic (Hypo; 14 days, 10.5% O(2)) adult rats. In addition, the mRNA expression of hypoxia-inducible factor (HIF)-1alpha and the protein expression of endothelial nitric oxide synthase (eNOS) were measured. Postischemic left ventricular function recovered to 66 +/- 6% and 67 +/- 5% of baseline in Norm and Hypo, respectively. In contrast, postischemic right ventricular function was 93 +/- 2% of baseline in Hypo vs. 67 +/- 3% in Norm (P < 0.05). Improved postischemic right ventricular function in Hypo (93 +/- 2% and 96 +/- 2% of baseline) was observed with 95% O(2) or 21% O(2) in the perfusate, and it was not attenuated by glibenclamide (5 and 10 micromol/l) (86 +/- 4% and 106 +/- 6% recovery). HIF-1alpha mRNA and eNOS protein expression were increased in both left and right hypoxic ventricles. In conclusion, postischemic right, but not left, ventricular function was improved by preceding chronic hypoxia. ATP-sensitive K(+) channels are not responsible for the increased right ventricular tolerance to ischemia after chronic hypoxia in adult rat hearts.

In hearts with chronic left ventricular (LV) systolic dysfunction secondary to hypertension or myocardial infarction, MAPK phosphorylation and/or activity are increased. Whether other settings of LV dysfunction not associated with ischemia-reperfusion are also characterized by increased MAPK phosphorylation or activity is unknown. After 3 wk of rapid LV pacing (400 beats/min), eight rabbits displayed clinical signs of heart failure (HF), and echocardiography revealed an increase in LV end-diastolic diameter from 15.6 +/- 0.7 (means +/- SE) to 18.8 +/- 0.7 mm and a reduced shortening fraction from 31 +/- 1to10 +/- 2% (both P < 0.05). Morphological alterations in HF included increased numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cardiomyocytes, extent of fibrosis, and cross-sectional cardiomyocyte area. Total p38 MAPK did not differ between failing and normal hearts (n = 8). However, p38 MAPK phosphorylation [164,488 +/- 29,323 vs. 43,565 +/- 14,817 arbitrary units (AU), P < 0.05, densitometry] and the activities of p38 MAPK-alpha and -beta were increased in failing compared with normal hearts (149,441 +/- 38,381 and 170,430 +/- 32,952 vs. 68,815 +/- 28,984 and 81,788 +/- 22,774 AU, respectively, both P < 0.05). In failing compared with normal hearts, total and phosphorylated JNK46 and JNK54 MAPK were increased, whereas total and phosphorylated ERK MAPK remained unchanged. In pacing-induced HF, p38 and JNK MAPK phosphorylation as well as p38 MAPK activity was increased. Further studies will have to define whether or not chronic specific blockade of MAPK activity can interfere with apoptosis/fibrosis and thereby attenuate the progression of HF.

Nitric oxide (NO) is involved in the control of myocardial metabolism. In normoperfused myocardium, NO synthase inhibition shifts myocardial metabolism from free fatty acid (FFA) toward carbohydrate utilization. Ischemic myocardium is characterized by a similar shift toward preferential carbohydrate utilization, although NO synthesis is increased. The importance of NO for myocardial metabolism during ischemia has not been analyzed in detail. We therefore assessed the influence of NO synthase inhibition with N(G)-nitro-l-arginine (l-NNA) on myocardial metabolism during moderate ischemia in anesthetized pigs. In control animals, the increase in left ventricular pressure with l-NNA was mimicked by aortic constriction. Before ischemia, l-NNA decreased myocardial FFA consumption (MV(FFA); P < 0.05), while consumption of carbohydrate and O(2) (MVo(2)) remained constant. ATP equivalents [calculated with the assumption of complete oxidative substrate decomposition (ATP(eq))] decreased with l-NNA (P < 0.05), associated with a decrease of regional myocardial function (P < 0.05). In contrast, aortic constriction had no effect on MV(FFA), while MVo(2) increased (P < 0.05) and ATP(eq) and regional myocardial function remained constant. During ischemia, alterations in myocardial metabolism were similar in control and l-NNA-treated animals: MV(FFA) decreased (P < 0.05) and net lactate consumption was reversed to net lactate production (P < 0.05). Regional myocardial function was decreased (P < 0.05), although more markedly in animals receiving l-NNA (P < 0.05). We conclude that the efficiency of oxidative metabolism was impaired by l-NNA per se, paralleled by impaired regional myocardial function. During ischemia, l-NNA had no effect on myocardial substrate consumption, indicating that NO synthases were no longer effectively involved in the control of myocardial metabolism.

B-type natriuretic peptide (BNP) has been reported to be released from the myocardium during ischemia. We hypothesized that BNP mediates cardioprotection during ischemia-reperfusion and examined whether exogenous BNP limits myocardial infarction and the potential role of ATP-sensitive potassium (K(ATP)) channel opening. Langendorff-perfused rat hearts underwent 35 min of left coronary artery occlusion and 120 min of reperfusion. The control infarct-to-risk ratio was 44.8 +/- 4.4% (means +/- SE). BNP perfused 10 min before ischemia limited infarct size in a concentration-dependent manner, with maximal protection observed at 10(-8) M (infarct-to-risk ratio: 20.1 +/- 5.2%, P < 0.01 vs. control), associated with a 2.5-fold elevation of myocardial cGMP above the control value. To examine the role of K(ATP) channel opening, glibenclamide (10(-6) M), 5-hydroxydecanoate (5-HD; 10(-4) M), or HMR-1098 (10(-5) M) was coperfused with BNP (10(-8) M). Protection afforded by BNP was abolished by glibenclamide or 5-HD but not by HMR-1098, suggesting the involvement of putative mitochondrial but not sarcolemmal K(ATP) channel opening. We conclude that natriuretic peptide/cGMP/K(ATP) channel signaling may constitute an important injury-limiting mechanism in myocardium.

Pretreatment with tumor necrosis factor-alpha (TNF-alpha) antibodies abolishes myocardial infarct size reduction by late ischemic preconditioning (IP). Whether or not TNF-alpha is also important for myocardial infarct size reduction by classic IP is unknown. Anesthetized rabbits were untreated (group 1, n = 7), classically preconditioned by 5 min left coronary artery occlusion/10 min reperfusion (group 2, n = 6), or pretreated with TNF-alpha antibodies without (group 3, n = 6) or with IP (group 4, n = 6) before undergoing 30 min of occlusion and 180 min of reperfusion. Infarct size in group 1 was 44 +/- 11 (means +/- SD)% of the area at risk. With a comparable area at risk, infarct size was reduced to 13 +/- 7%, 23 +/- 8%, and 19 +/- 12% (all P < 0.05) in groups 2, 3, and 4, respectively. The circulating TNF-alpha concentration was increased during ischemia in group 1 from 752 +/- 403 to 1,542 +/- 482 U/ml (P < 0.05) but remained unchanged in all other groups. Circulating TNF-alpha concentration during ischemia and infarct size correlated in all groups (r = 0.76). IP, TNF-alpha antibodies, and the combined approach reduced infarct size to a comparable extent. Therefore, the question of whether or not TNF-alpha is causally involved in the infarct size reduction by IP in rabbits could not be answered.

Free oxyradicals are involved in the signal transduction of ischemic preconditioning in rats and rabbits. Data from larger mammals in which the infarct development is closer to that in humans are lacking. We have therefore investigated the impact of the radical scavenger ascorbic acid on ischemic preconditioning in pigs. In 33 anesthetized pigs, the left anterior descending coronary artery was perfused from an extracorporeal circuit. Infarct size (measured as percent area at risk) was determined by triphenyltetrazolium chloride staining. In placebo-treated animals undergoing 90 min of severe ischemia and 120 min of reperfusion, infarct size averaged 26.9 +/- 3.9% (mean +/- SE; n = 9). Ischemic preconditioning by 10 min of ischemia and 15 min of reperfusion reduced infarct size to 6.4 +/- 2.4% (P < 0.05 vs. placebo; n = 9). Intravenous infusion of ascorbic acid (30 min before ischemic preconditioning or ischemia; 2-g bolus followed by 25 mg/min until the end of ischemia) had no effect on infarct size per se (22.6 +/- 6.5%; n = 6), but largely abolished the infarct size reduction by ischemic preconditioning (19.1 +/- 5.4%; n = 9). Scavenging of free oxyradicals with ascorbic acid largely attenuates the beneficial effect of ischemic preconditioning in pigs.

The effect of synthetic parathyroid hormone (PTH)-related peptide [PTHrP(1-34)] on regional myocardial function was studied in 11 anesthetized pigs. Intracoronary infusion of PTHrP (cumulative dose: 14 +/- 1 microg) decreased coronary resistance to 33 +/- 2% of baseline (P < 0.05) and regional myocardial function to 90 +/- 3% of baseline (not significant). Ischemia-reperfusion alters the activity of several kinases and therefore possibly the myocardial effects of PTHrP. In stunned myocardium, induced by 20-min ischemia and 30-min reperfusion, the dose of PTHrP reducing coronary resistance to a minimum of 29 +/- 2% was decreased to 8 +/- 2 microg (P < 0.05). Regional myocardial function was no longer decreased but increased to 132 +/- 9% (P < 0.05). The increase in regional myocardial function during PTHrP was inversely related to baseline function at 30-min reperfusion in vivo (r = 0.9) as well as in myocytes isolated from stunned pig hearts (r = 0.7). In isolated rat hearts subjected to 30-min global ischemia followed by 30-min reperfusion, blockade of endogenous PTHrP by d-Trp(12)-Tyr(34)-PTH(7-34) attenuated the recovery of left ventricular developed pressure by 30 +/- 14% (P < 0.05). Thus endogenous and exogenous PTHrP impact on the function of stunned myocardium.

Protein kinase Cepsilon (PKCepsilon) plays a central role in ischemic preconditioning (IP) in mice and rabbits, and activated PKCepsilon colocalizes with and phosphorylates connexin43 (Cx43) in rats and humans. Whether or not Cx43 contributes to the mechanism(s) of IP in vivo is yet unknown. Therefore, wild-type (n = 8) and heterozygous Cx43-deficient mice (n = 8) were subjected to 30 min occlusion and 120 min reperfusion of the left anterior descending coronary artery. IP was induced by one cycle of 5 min occlusion and 10 min reperfusion (n = 8/8 mice) before the sustained occlusion. Infarct size was reduced by IP in wild-type mice [11.3 +/- 3.4% vs. 23.7 +/- 7.2% of the left ventricle (LV), P < 0.05] but not in Cx43-deficient mice (26.0 +/- 6.0% vs. 25.1 +/- 3.8% of LV). Also, three cycles of 5 min occlusion and 10 min reperfusion (n = 5) did not induce protection in Cx43-deficient mice (27.6 +/- 5.5 % of LV). Thus Cx43 contributes to the protection of IP in mice in vivo.

10.1152/ajpheart.00925. 2001.-Parathyroid hormone-related peptide (PTHrP) is expressed throughout the cardiovascular system and is able to dilate vessels. This study investigated whether mechanical forces generated by changes in regional perfusion influence PTHrP release from the coronary vascular bed. Experiments were performed in vitro on saline-perfused rat hearts or isolated coronary endothelial cells exposed to cyclic strain and in vivo in anesthetized pigs. In vitro, PTHrP release from saline-perfused rat hearts was strongly correlated with coronary flow (r = 0.84). Increasing coronary flow from 5 to 10 ml/min increased PTHrP release from 442 +/- 42 to 1,563 +/- 167 pg/min. Increasing the viscosity of the perfusate did not change basal PTHrP release. Increasing flow without a concomitant increase in pressure did not lead to an increase in release rate, but reduction in pressure under flow-constant conditions reduced PTHrP release rate. Cyclic strain induced a strain-dependent release of PTHrP from endothelial cells that was inhibited by the addition of a calcium-chelating agent. In vivo, there was a net release of PTHrP in the coronary circulation and decreases in coronary flow and pressure decreased the PTHrP release rate. Bradykinin in the presence of constant pressure increased PTHrP release, probably by increasing the intracellular calcium concentration in coronary endothelial cells. In summary, mechanical forces evoked by blood flow can trigger a constant PTHrP release.

Free radicals have been implicated in the etiology of cardiac dysfunction during sepsis, but the actual species responsible remains unclear. We studied the alterations in myocardial nitric oxide (NO), superoxide, and peroxynitrite generation along with cardiac mechanical function and efficiency in hearts from lipopolysaccharide (LPS)-treated rats. Six hours after LPS (4 mg/kg ip) or saline (control) treatment, hearts were isolated and perfused for 1 h with recirculating Krebs-Henseleit buffer and paced at 300 beats/min. Cardiac work, O(2) consumption, and cardiac efficiency were markedly depressed in LPS hearts compared with controls. Plasma nitrate/nitrite level was elevated in LPS rats, and ventricular NO production was enhanced as measured by electron spin resonance spectroscopy, Ca(2+)-independent NO synthase (NOS) activity, and inducible NOS immunohistochemistry. Ventricular superoxide production was also enhanced in LPS-treated hearts as seen by lucigenin chemiluminescence and xanthine oxidase activity. Increased nitrotyrosine staining (immunohistochemistry) and higher lipid hydroperoxides levels were also detected in LPS-treated hearts, indicating oxygen radical-induced stress. Enhanced generation of both NO and superoxide, and thus peroxynitrite, occur in dysfunctional hearts from endotoxemic rats.

Clinical studies have suggested that long-term nitrate treatment does not improve and may even worsen cardiovascular mortality, and the possible role of nitrate tolerance has been suspected. Nitrate tolerance has been recently shown to increase vascular superoxide and peroxynitrite production leading to vascular dysfunction. Nevertheless, nitrates exert direct cardiac effects independent from their vascular actions. Therefore, we investigated whether in vivo nitroglycerin treatment leading to vascular nitrate tolerance increases cardiac formation of nitric oxide (NO), reactive oxygen species, and peroxynitrite, thereby leading to cardiac dysfunction. Nitrate tolerance increased bioavailability of NO in the heart without increasing formation of reactive oxygen species. Despite elevated myocardial NO, neither cardiac markers of peroxynitrite formation nor cardiac mechanical function were affected by nitroglycerin treatment. However, serum free nitrotyrosine, a marker for systemic peroxynitrite formation, was significantly elevated in nitroglycerin-treated animals. This is the first demonstration that, although the systemic effects of nitroglycerin may be deleterious due to enhancement of extracardiac peroxynitrite formation, nitroglycerin does not result in oxidative damage in the heart.

Microembolized myocardium is characterized by perfusion-contraction mismatch with reduced contractile function and unchanged or even elevated blood flow. The present study investigated the consequences of microembolization on coronary and inotropic reserves. In eight anesthetized dogs, left circumflex coronary blood flow (CBF), regional blood flow (RBF), and posterior systolic wall thickening were measured. Repetitive injection of 42-microm microspheres into the left circumflex coronary artery decreased systolic wall thickening by 50% (17.2 +/- 2.4% vs. 8.0 +/- 1.4%; means +/- SD). Coronary reserve was determined by either intracoronary infusion of adenosine (n = 4) or the reactive hyperemia response following 15 s of coronary occlusion (n = 4); inotropic reserve was recruited by intracoronary infusion of dobutamine. The amount of injected microspheres was 158,000 +/- 48,000. CBF (45.5 +/- 16.5 vs. 47.8 +/- 14.4 ml/min) and RBF (1.15 +/- 0.18 vs. 1.33 +/- 0.39 ml x min(-1) x g(-1)) remained unchanged. Coronary reserve in response to intracoronary infusion of adenosine (410 +/- 94% vs. 290 +/- 77%; P < 0.05) and reactive hyperemia repayment (360 +/- 174% vs. 155 +/- 66%; P < 0.05) were blunted after microembolization. Inotropic reserve, i.e., the increment in systolic wall thickening with dobutamine, was decreased from 12.4 +/- 3.9% to 8.0 +/- 3.3% (P < 0.05). We conclude that coronary microembolization reduces coronary and inotropic reserves.

We tested whether or not endogenous nitric oxide (NO) attenuates beta-adrenergic inotropic responsiveness during normoperfusion or moderate myocardial ischemia. In 13 anesthetized pigs with a cannulated left anterior descending (LAD) coronary artery, the maximal contractile responses to intracoronary dobutamine and calcium were assessed during normoperfusion and at the end of a 90-min period of moderate ischemia (50% reduction in coronary arterial inflow) without (group 1, n = 6) and with (group 2, n = 7) prior inhibition of NO synthesis [30 mg/kg iv N(omega)-nitro-L-arginine (L-NNA)]. Contractile function was assessed by a regional work index (sonomicrometry, micromanometry, mm. mmHg). In groups 1 and 2 during normoperfusion, the maximal increase of the work index was greater with calcium than with dobutamine. At the end of ischemia in group 1, the baseline work index was decreased by approximately 50%, and the subsequent maximal increase of the work index with dobutamine, but not with calcium, was reduced compared with normoperfusion. In group 2 during normoperfusion, L-NNA did not alter the maximal increases of the work index with dobutamine or calcium. At the end of ischemia, the baseline work index was reduced by 64%, and the subsequent maximal increases of the work index with both dobutamine and calcium were reduced compared with normoperfusion; however, the response to calcium was still greater than that to dobutamine. We conclude that endogenous NO does not limit beta-adrenergic inotropic responsiveness in normoperfused or moderately ischemic porcine myocardium.

It is unclear whether perfusion-contraction matching (PCM) is maintained during prolonged myocardial ischemia. In 27 anesthetized pigs, left anterior descending coronary arterial inflow was reduced to decrease an anterior work index (WI) at 5 min of hypoperfusion by 40% and then maintained at this level for 12 or 24 h. With 12 h of hypoperfusion, the myocardium remained viable in 6 of 7 pigs (with triphenyltetrazolium chloride; TTC) and with 24 h of hypoperfusion in 5 of 11 pigs (TTC, histology). The reduction in WI to 62 +/- 4 and 62 +/- 3% of baseline in the two groups was matched to the reduction of transmural blood flow (TBF; microspheres) at 5 min of hypoperfusion, averaging 59 +/- 4 and 60 +/- 2% of baseline. With prolonged hypoperfusion, WI decreased to 30 +/- 5% at 12 h and 18 +/- 3% at 24 h; TBF remained unchanged (53 +/- 4 and 54 +/- 4%). The added calcium concentration required for the half-maximal increase in WI increased from 121 +/- 25 microg/ml blood at baseline to 192 +/- 26 microg/ml blood at 12 h of hypoperfusion. Thus, with hypoperfusion for 24 h, PCM is progressively lost, and calcium responsiveness is reduced.

Endogenous opioids are involved in ischemic preconditioning (IP) in several species. Whether or not opioids are important for IP and short-term myocardial hibernation (STMH) in pigs is currently unknown. In 34 enflurane-anesthetized pigs, the left anterior descending coronary artery was flow constantly perfused. Subendocardial blood flow (Endo), infarct size (IS; percent area at risk), and the free energy change of ATP hydrolysis (DeltaG) were determined. After 90-min severe ischemia and 120-min reperfusion, IS averaged 28.3 +/- 5.4% (means +/- SE) (n = 8; Endo: 0.047 +/- 0.009 ml. min(-1) x g(-1)). IP by 10-min ischemia and 15-min reperfusion reduced IS to 9.9 +/- 3.8% (P < 0.05, n = 8; Endo: 0.044 +/- 0.009 ml. min(-1) x g(-1)). After naloxone (1 mg/kg iv followed by 2 microg x kg(-1) x min(-1)), IS averaged 25.8 +/- 7.0% (n = 6; Endo: 0.039 +/- 0.008 ml x min(-1) x g(-1)) without and 24.7 +/- 4.7% (n = 6; Endo: 0.044 +/- 0.006 ml x min(-1) x g(-1)) with IP. At 5-min moderate ischemia in the presence of naloxone, Endo decreased from 0.90 +/- 0.07 to 0.28 +/- 0.03 ml x min(-1) x g(-1)and DeltaG decreased from -58.6 +/- 1.0 to -52.6 +/- 0.4 kJ/mol. Prolongation of ischemia to 90 min did not alter Endo, but DeltaG recovered toward control values (57.7 +/- 1.1 kJ/mol), and the myocardium remained viable. These responses are identical to those of nonnaloxone-treated pigs. Endogenous opioids are involved in IP but not in STMH in pigs.

Ischemic preconditioning (IP) protects the rat liver. In pigs, in which hepatic tolerance to ischemia is similar to that in humans, information on IP is lacking. Therefore, in enflurane-anesthetized pigs, hepatic vessels were occluded for 120 min (protocol 1) or 200 min (protocol 2) without (control) and with IP (3 times 10 min ischemia-reperfusion each). In protocol 1, cumulative bile flow (CBF) during reperfusion was greater in IP (47.3 +/- 5.2 ml/8 h) than in control (17.1 +/- 7.8 ml/8 h, P < 0.05). ATP content tended to recover toward normal during reperfusion in IP, whereas it remained at ischemic levels in control. Serum enzyme concentrations increased similarly during reperfusion, and <1% hepatocytes were necrotic or stained terminal deosynucleotidyl transferase-mediated dUTP nick-end labeling-positive in control and IP groups. In protocol 2, no differences in CBF, ATP, or serum enzyme concentrations during reperfusion were measured between control and IP groups, except for a somewhat reduced lactate dehydrogenase in IP. The number of necrotic or terminal deosynucleotidyl transferase-mediated dUTP nick-end labeling-positive hepatocytes tended to be greater in the IP than the control group. Thus IP provides some functional protection against reversible ischemia but no protection during prolonged ischemia in pigs.

A close relationship exists between regional myocardial blood flow (RMBF) and function during acute coronary inflow restriction (perfusion-contraction matching). However, the relationship of flow and function during coronary microvascular obstruction is unknown. In 12 anesthetized dogs, the left circumflex coronary artery was perfused from an extracorporeal circuit. After control measurements, 3,000 microspheres (42 micrometer diameter) per milliliter per minute inflow were injected to cause a microembolism (ME, n = 6). With unchanged systemic hemodynamics and RMBF, posterior systolic wall thickening (PWT) decreased from 19.8 +/- 1.9% SD at control to 13.3 +/- 4.0, 10.3 +/- 3.8, and 6.9 +/- 4.7% (P < 0.05 vs. control) at 1, 4, and 8 h, respectively. For comparison, inflow was progressively reduced to match PWT to that of the ME group at 1, 4, and 8 h (stenosis, STE, n = 6). RMBF in the STE group was reduced in proportion to PWT. Infarct size was not different among groups (6.5 +/- 4.5 vs. 3.4 +/- 3.2%). However, the number of leukocytes infiltrating the area at risk was significantly greater in the ME group than in the STE group. Coronary microembolization results in perfusion-contraction mismatch and is associated with an inflammatory response.

Exercise enhances cardiac output and blood flow to working skeletal muscles but decreases visceral perfusion. The alterations in nitric oxide synthase (NOS) activity and/or expression of the cardiopulmonary, skeletal muscle, and visceral organs induced by swim training are unknown. In sedentary and swim-trained rats (60 min twice/day for 3-4 wk), we studied the alterations in NOS in different tissues along with hindquarter vasoreactivity in vivo during rest and mesenteric vascular bed reactivity in vitro. Hindquarter blood flow and conductance were reduced by norepinephrine in both groups to a similar degree, whereas N(G)-nitro-L-arginine methyl ester reduced both indexes to a greater extent in swim-trained rats. Vasodilator responses to ACh, but not bradykinin or S-nitroso-N-acetyl-penicillamine, were increased in swim-trained rats. Ca(2+)-dependent NOS activity was enhanced in the hindquarter skeletal muscle, lung, aorta, and atria of swim-trained rats together with increased expression of neuronal NOS in the hindquarter skeletal muscle and endothelial NOS in the cardiopulmonary organs. Mesenteric arterial bed vasoreactivity was unaltered by swim training. Physiological adaptations to swim training are characterized by enhanced hindquarter ACh-induced vasodilation with upregulation of neuronal NOS in skeletal muscle and endothelial NOS in the lung, atria, and aorta.

The importance of the activation of mitogen-activated protein kinases (MAPK) for the cardioprotection achieved by ischemic preconditioning (IP) is still controversial. We therefore measured infarct size and p38, extracellular signal-regulated kinase (ERK), and c-Jun NH(2)-terminal kinase (JNK) MAPK phosphorylation (by biopsies) in enflurane-anesthetized pigs. After 90 min low-flow ischemia and 120 min reperfusion, infarct size averaged 18.3 +/- 12.4 (SD)% (group 1, n = 14). At similar subendocardial blood flows, IP by 10 min ischemia and 15 min reperfusion (group 2, n = 14) reduced infarct size to 6.2 +/- 5.1% (P < 0.05). An inconsistent increase in p38, ERK, and p54 JNK phosphorylation (by Western blot) was found during IP; p46 JNK phosphorylation increased with the subsequent reperfusion. At 8 min of the sustained ischemia, p38, ERK, and p54 JNK phosphorylation were increased with no difference between groups (medians: p38: 207% of baseline in group 1 vs. 153% in group 2; ERK: 142 vs. 144%; p54 JNK: 171 vs. 155%, respectively). MAPK phosphorylation and reduction of infarct size by IP were not correlated, thus not supporting the concept of a causal role of MAPK in mediating cardioprotection by IP.

Short-term hibernating myocardium is characterized by reduced contractile function during persistent moderate ischemia, the recovery of metabolic parameters, and the absence of necrosis. To study the afterload dependence of regional wall excursion in short-term hibernating myocardium, in 11 enflurane-anesthetized swine the left anterior descending coronary artery was cannulated and hypoperfused for 90 min to reduce anterior systolic wall thickening (WT, sonomicrometry) by 60%. Under control conditions, at 5 and 90 min ischemia the descending thoracic aorta was acutely constricted to increase left ventricular (LV) pressure by 30 mmHg. Under control conditions, increased LV pressure resulted in decreased WT [i.e., a negative slope of the relationship between WT and LV end-systolic pressure: -11.2 +/- 4.2 (SD) microm/mmHg]. This slope was further significantly decreased at 5 min ischemia (-26.5 +/- 8.8 microm/mmHg) but returned toward control values in short-term hibernating myocardium at 90 min ischemia (-17.2 +/- 6.6 microm/mmHg). At 30 min reperfusion, the slope was once more significantly decreased (-27.8 +/- 8.1 microm/mmHg). In conclusion, WT in short-term hibernating myocardium is less afterload dependent than in acutely ischemic and reperfused myocardium.