Among the numerous mechanisms proposed for myocardial stunning, three appear to be more plausible: 1) generation of oxygen radicals, 2) calcium overload, and 3) excitation-contraction uncoupling. ...First, the evidence for a pathogenetic role of oxygen-derived free radicals in myocardial stunning is overwhelming. In the setting of a single 15-minute coronary occlusion, mitigation of stunning by antioxidants has been reproducibly observed by several independent laboratories. Similar protection has been recently demonstrated in the conscious animal, that is, in the most physiological experimental preparation available. Furthermore, generation of free radicals in the stunned myocardium has been directly demonstrated by spin trapping techniques, and attenuation of free radical generation has been repeatedly shown to result in attenuation of contractile dysfunction. Numerous observations suggest that oxyradicals also contribute to stunning in other settings: after global ischemia in vitro, after global ischemia during cardioplegic arrest in vivo, and after multiple brief episodes of regional ischemia in vivo. Compelling evidence indicates that the critical free radical damage occurs in the initial moments of reflow, so that myocardial stunning can be viewed as a sublethal form of oxyradical-mediated "reperfusion injury." Second, there is also considerable evidence that a transient calcium overload during early reperfusion contributes to postischemic dysfunction in vitro; however, the importance of this mechanism in vivo remains to be defined. Third, inadequate release of calcium by the sarcoplasmic reticulum, with consequent excitation-contraction uncoupling, may occur after multiple brief episodes of regional ischemia, but its role in other forms of postischemic dysfunction has not been explored. It is probable that multiple mechanisms contribute to the pathogenesis of myocardial stunning. The three hypotheses outlined above are not mutually exclusive and in fact may represent different steps of the same pathophysiological cascade. Thus, generation of oxyradicals may cause sarcoplasmic reticulum dysfunction, and both of these processes may lead to calcium overload, which in turn could exacerbate the damage initiated by oxygen species. The concepts discussed in this review should provide not only a conceptual framework for further investigation of the pathophysiology of reversible ischemia-reperfusion injury but also a rationale for developing clinically applicable interventions designed to prevent postischemic ventricular dysfunction.
The goal of this study was to determine the role of the Janus tyrosine kinase (JAK)-signal transducers and activators of transcription (STAT) pathway in the late phase of ischemic preconditioning ...(PC). A total of 230 mice were used. At 5 min after ischemic PC (induced with six cycles of 4-min coronary occlusion/4-min reperfusion), immunoprecipitation with anti-phosphotyrosine (anti-pTyr) antibodies followed by immunoblotting with anti-JAK antibodies revealed increased tyrosine phosphorylation of JAK1 (+257 ± 53%) and JAK2 (+238 ± 35%), indicating rapid activation of these two kinases. Similar results were obtained by immunoblotting with anti-pTyr-JAK1 and anti-pTyr-JAK2 antibodies. Western analysis with anti-pTyr-STAT antibodies demonstrated a marked increase in nuclear pTyr-STAT1 (+301 ± 61%) and pTyr-STAT3 (+253 ± 60%) 30 min after ischemic PC, which was associated with redistribution of STAT1 and STAT3 from the cytosolic to the nuclear fraction and with an increase in STAT1 and STAT3 γ-IFN activation site DNA-binding activity (+606 ± 64%), indicating activation of STAT1 and STAT3. No nuclear translocation or tyrosine phosphorylation of STAT2, STAT4, STAT5A, STAT5B, or STAT6 was observed. Pretreatment with the JAK inhibitor AG-490 20 min before the six occlusion/reperfusion cycles blocked the enhanced tyrosine phosphorylation of JAK1 and JAK2 and the increased tyrosine phosphorylation, nuclear translocation, and enhanced DNA-binding activity of STAT1 and STAT3. The same dose of AG-490 abrogated the protection against myocardial infarction and the concomitant up-regulation of inducible NO synthase (iNOS) protein and activity observed 24 h after ischemic PC. Taken together, these results demonstrate that ischemic PC induces isoform-selective activation of JAK1, JAK2, STAT1, and STAT3, and that ablation of this response impedes the up-regulation of iNOS and the concurrent acquisition of ischemic tolerance. This study demonstrates that the JAK-STAT pathway plays an essential role in the development of late PC. The results reveal a signaling mechanism that underlies the transcriptional up-regulation of the cardiac iNOS gene and the adaptation of the heart to ischemic stress.
Stem/progenitor cells are usually cultured at atmospheric O
2
tension (21%); however, since physiologic O
2
tension in the heart is ∼5%, using 21% O
2
may cause oxidative stress and toxicity. Cardiac ...mesenchymal cells (CMCs), a newly discovered and promising type of progenitor cells, are effective in improving left ventricle (LV) function after myocardial infarction (MI). We have previously shown that, compared with 21% O
2
, culture at 5% O
2
increases CMC proliferation, telomerase activity, telomere length, and resistance to severe hypoxia
in vitro
. However, it is unknown whether these beneficial effects of 5% O
2
in vitro
translate into greater therapeutic efficacy
in vivo
in the treatment of heart failure. Thus, murine CMCs were cultured at 21% or 5% O
2
. Mice with heart failure caused by a 60-min coronary occlusion followed by 30 days of reperfusion received vehicle, 21% or 5% O
2
CMCs via echocardiography-guided intraventricular injection. After 35 days, the improvement in LV ejection fraction effected by 5% O
2
CMCs was > 3 times greater than that afforded by 21% O
2
CMCs (5.2 vs. 1.5 units,
P
< 0.01). Hemodynamic studies (Millar catheter) yielded similar results both for load-dependent (LV dP/dt) and load-independent (end-systolic elastance) indices. Thus, two independent approaches (echo and hemodynamics) demonstrated the therapeutic superiority of 5% O
2
CMCs. Further, 5% O
2
CMCs, but not 21% O
2
CMCs, significantly decreased scar size, increased viable myocardium, reduced LV hypertrophy and dilatation, and limited myocardial fibrosis both in the risk and non-infarcted regions. Taken together, these results show, for the first time, that culturing CMCs at physiologic (5%) O
2
tension provides superior therapeutic efficacy in promoting cardiac repair
in vivo
. This concept may enhance the therapeutic potential of CMCs. Further, culture at 5% O
2
enables greater numbers of cells to be produced in a shorter time, thereby reducing costs and effort and limiting cell senescence. Thus, the present study has potentially vast implications for the field of cell therapy.
Echocardiography is an essential diagnostic tool for accurate noninvasive assessment of cardiac structure and function in vivo. However, the use of anesthetic agents during echocardiographic studies ...is associated with alterations in cardiac anatomical and functional parameters. We sought to systematically compare the effects of three commonly used anesthetic agents on echocardiographic measurements of left ventricular (LV) systolic and diastolic function, LV dimensions, and LV mass in rats. Adult male Fischer 344 rats underwent echocardiographic studies under pentobarbital (PB, 25 mg/kg i.p.) (group I, n = 25), inhaled isoflurane (ISF, 1.5%) (group II, n = 25),or ketamine/xylazine (K/X, 37 mg/kg ketamine and 7 mg/kg xylazine i.p.) (group III, n = 25) anesthesia in a cross-over design. Echocardiography was also performed in an additional group of unanesthetized conscious rats (group IV, n = 5). Postmortem studies were performed to validate echocardiographic assessment of LV dimension and mass. Rats in group I exhibited significantly higher LV ejection fraction, fractional shortening, fractional area change, velocity of circumferential fiber shortening corrected for heart rate, and heart rate as compared with groups II and III. LV end-diastolic volume, end-diastolic diameter, and cross-sectional area in diastole were significantly smaller in group I compared with groups II and III. Cardiac output was significantly lower in group III compared with groups I and II. Postmortem LV mass measurements correlated well with echocardiographic estimation of LV mass for all anesthetic agents, and the correlation was best with PB anesthesia. Limited echocardiographic data obtained in conscious rats were similar to those obtained under PB anesthesia. We conclude that compared with ISF and K/X anesthesia, PB anesthesia at a lower dose yields echocardiographic LV structural and functional data similar to those obtained in conscious rats. In addition, PB anesthesia also facilitates more accurate estimation of LV mass.
Unlike the early phase of preconditioning (PC), which lasts 2 to 3 hours and protects against infarction but not against stunning, the late phase of PC lasts 3 to 4 days and protects against both ...infarction and stunning, suggesting that it may have greater clinical relevance. It is now clear that late PC is a polygenic phenomenon that requires the simultaneous activation of multiple stress-responsive genes. Chemical signals released by a sublethal ischemic stress (such as NO, reactive oxygen species, and adenosine) trigger a complex cascade of signaling events that includes the activation of protein kinase C, Src protein tyrosine kinases, and nuclear factor kappaB and culminates in increased synthesis of inducible NO synthase, cyclooxygenase-2, aldose reductase, Mn superoxide dismutase, and probably other cardioprotective proteins. An analogous sequence of events can be triggered by a variety of stimuli, such as heat stress, exercise, and cytokines. Thus, late PC appears to be a universal response of the heart to stress in general. Importantly, the cardioprotective effects of late PC can be reproduced pharmacologically with clinically relevant agents (eg, NO donors, adenosine receptor agonists, endotoxin derivatives, or opioid receptor agonists), suggesting that this phenomenon might be exploited for therapeutic purposes. The purpose of this review is to summarize current information regarding the pathophysiology and mechanism of late PC.
Over the past decade, an enormous number of studies (>100) have focused on the role of nitric oxide (NO) in myocardial ischemia. It is important to distinguish the function of NO in unstressed ...(non-preconditioned) myocardium from its function in preconditioned myocardium (i.e. myocardium that has shifted to a defensive phenotype in response to stress). Of the 92 studies that have examined the role of NO in modulating the severity of ischemia/reperfusion injury in non-preconditioned myocardium, the vast majority 67 (73%) have concluded that NO (either endogenous or exogenous) has a protective effect and only 11 (12%) found a detrimental effect. The proportion of studies supporting a cytoprotective role of NO is similar in vivo35 (71%) out of 49 and in vitro32 (74%) out of 43. With regard to the delayed acquisition of tolerance to ischemia late preconditioning (PC), overwhelming evidence indicates a critical role of NO in this phenomenon. Specifically, enhanced biosynthesis of NO by eNOS is essential to trigger the late phase of ischemia-induced and exercise-induced PC, and enhanced NO production by iNOS is obligatorily required to mediate the anti-stunning and anti-infarct actions of late PC elicited by five different stimuli (ischemia, adenosine A1agonists, opioidδ1 agonists, endotoxin derivatives and exercise). Thus, NO plays a dual role in the pathophysiology of the late phase of PC, acting initially as the trigger and subsequently as the mediator of this adaptive response (“NO hypothesis of late PC”). The diversity of the PC stimuli that converge on iNOS implies that the upregulation of this enzyme is a central mechanism whereby the myocardium protects itself from ischemia. The NO hypothesis of late PC has thus revealed a cytoprotective function of iNOS in the heart, a novel paradigm which has recently been extended to other tissues, including kidney and intestine. Other corollaries of this hypothesis are that the heart responds to stress in a biphasic manner, utilizing eNOS as an immediate but short-term response and iNOS as a delayed but long-term defense, and that the fundamental difference between non-preconditioned and late preconditioned myocardium is the tissue level of iNOS-derived NO, which is tonically higher in the latter compared with the former. Hence, late PC can be viewed as a state of enhanced NO synthesis. The NO hypothesis of late PC has important therapeutic implications. In experimental animals, administration of NO donors in lieu of ischemia can faithfully reproduce the molecular and functional aspects of ischemia-induced late PC, indicating that NO is not only necessary but also sufficient to induce late PC. The recent demonstration that nitroglycerin also induces late PC in patients provides proof-of-principle for the concept that nitrates could be used as a PC-mimetic therapy for the prophylaxis of ischemic injury in the clinical arena. This novel application of nitrates could be as important as, or perhaps even more important than, their current use as antianginal and preload-reducing agents. In addition, gene transfer of either eNOS or iNOS has been shown to replicate the infarct-sparing actions of ischemic PC, suggesting that NOS gene therapy could be an effective strategy for alleviating ischemia/reperfusion injury. Ten years of research have demonstrated that NO plays a fundamental biological role in protecting the heart against ischemia/reperfusion injury. The time has come to translate this enormous body of experimental evidence into clinically useful therapies by harnessing the cytoprotective properties of NO.
Whether or not ROS are the actual initiator or a downstream mediator of stunning, the theory that they play a major causative role in this phenomenon is now a proven hypothesis. ROS play a prominent ...role in multiple forms of stunning. Furthermore, the role of ROS is prominent not only in open-chest models but also in conscious animal preparations. The major challenge for future studies will be to identify the specific molecular targets of ROS-mediated damage and to decipher the pathways whereby ROS and calcium interact in the genesis of myocardial stunning.
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