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BackgroundThe regenerative capacity of the heart after myocardial infarction is limited. Our previous study showed that ectopic introduction of 4 cell cycle factors (4F; CDK1 [cyclin-dependent kinase 1], CDK4 [cyclin-dependent kinase 4], CCNB [cyclin B1], and CCND [cyclin D1]) promotes cardiomyocyte proliferation in 15% to 20% of infected cardiomyocytes in vitro and in vivo and improves cardiac function after myocardial infarction in mice.MethodsUsing temporal single-cell RNA sequencing, we aimed to identify the necessary reprogramming stages during the forced cardiomyocyte proliferation with 4F on a single cell basis. Using rat and pig models of ischemic heart failure, we aimed to start the first preclinical testing to introduce 4F gene therapy as a candidate for the treatment of ischemia-induced heart failure.ResultsTemporal bulk and single-cell RNA sequencing and further biochemical validations of mature human induced pluripotent stem cell-derived cardiomyocytes treated with either LacZ or 4F adenoviruses revealed full cell cycle reprogramming in 15% of the cardiomyocyte population at 48 hours after infection with 4F, which was associated mainly with sarcomere disassembly and metabolic reprogramming (n=3/time point/group). Transient overexpression of 4F, specifically in cardiomyocytes, was achieved using a polycistronic nonintegrating lentivirus (NIL) encoding 4F; each is driven by a TNNT2 (cardiac troponin T isoform 2) promoter (TNNT2-4Fpolycistronic-NIL). TNNT2-4Fpolycistronic-NIL or control virus was injected intramyocardially 1 week after myocardial infarction in rats (n=10/group) or pigs (n=6-7/group). Four weeks after injection, TNNT2-4Fpolycistronic-NIL-treated animals showed significant improvement in left ventricular ejection fraction and scar size compared with the control virus-treated animals. At 4 months after treatment, rats that received TNNT2-4Fpolycistronic-NIL still showed a sustained improvement in cardiac function and no obvious development of cardiac arrhythmias or systemic tumorigenesis (n=10/group).ConclusionsThis study provides mechanistic insights into the process of forced cardiomyocyte proliferation and advances the clinical feasibility of this approach by minimizing the oncogenic potential of the cell cycle factors owing to the use of a novel transient and cardiomyocyte-specific viral construct.

The limited availability of human heart tissue and its complex cell composition are major limiting factors for the reliable testing of drug efficacy and toxicity. Recently, we developed functional human and pig heart slice biomimetic culture systems that preserve the viability and functionality of 300 μm heart slices for up to 6 days. Here, we tested the reliability of this culture system for testing the cardiotoxicity of anti-cancer drugs. We tested three anti-cancer drugs (doxorubicin, trastuzumab, and sunitinib) with known different mechanisms of cardiotoxicity at three concentrations and assessed the effect of these drugs on heart slice viability, structure, function and gene expression. Slices incubated with any of these drugs for 48 h showed diminished in viability as well as loss of cardiomyocyte structure and function. Mechanistically, RNA sequencing of doxorubicin-treated tissues demonstrated a significant downregulation of cardiac genes and upregulation of oxidative stress responses. Trastuzumab treatment downregulated cardiac muscle contraction-related genes consistent with its clinically known effect on cardiomyocytes. Interestingly, sunitinib treatment resulted in significant downregulation of angiogenesis-related genes, in line with its mechanism of action. Similar to hiPS-derived-cardiomyocytes, heart slices recapitulated the expected toxicity of doxorubicin and trastuzumab, however, slices were superior in detecting sunitinib cardiotoxicity and mechanism in the clinically relevant concentration range of 0.1-1 μM. These results indicate that heart slice culture models have the potential to become a reliable platform for testing and elucidating mechanisms of drug cardiotoxicity.

The falsification of data related to c-kit+ cardiac progenitor cells (CPCs) by a Harvard laboratory has been a veritable tragedy. Does this fraud mean that CPCs are not beneficial in models of ischemic cardiomyopathy? At least 50 studies from 26 laboratories independent of the Harvard group have reported beneficial effects of CPCs in mice, rats, pigs, and cats. The mechanism of action remains unclear. Our group has shown that CPCs do not engraft in the diseased heart, do not differentiate into new cardiac myocytes, do not regenerate dead myocardium, and thus work via paracrine mechanisms. A casualty of the misconduct at Harvard has been the SCIPIO trial, a collaboration between the Harvard group and the group in Louisville. The retraction of the SCIPIO paper was caused exclusively by issues with data generated at Harvard, not those generated in Louisville. In the retraction notice, the Lancet editors stated: 'Although we do not have any reservations about the clinical work in Louisville that used the preparations from Anversa's laboratory in good faith, the lack of reliability regarding the laboratory work at Harvard means that we are now retracting this paper'. We must be careful not to dismiss all work on CPCs because of one laboratory's misconduct. An unbiased review of the literature supports the therapeutic potential of CPCs for heart failure at the preclinical level. Key words: cardiac progenitor cell, c-kit, stem cells, cell therapy, chronic ischemic cardiomyopathy, myocardial infarction. La falsification de donnees liees aux cellules progenitrices cardiaques c-kit+ (CPC) par un laboratoire de Harvard a constitue une veritable tragedie. Cette fraude signifie-t-elle pour autant que les CPC ne contribuent pas aux avantages des modeles de cardiomyopathie ischemique? Au moins 50 etudes issues de 26 laboratoires independants dugroupe de Harvard ont rapporte des bienfaits obtenus avec les CPC chez la souris, le rat, le porc et le chat. Le mode d'action demeure a clarifier. Notre groupe a montre que les CPC ne se greffent pas au creur lese, ne se differencient pas en nouveaux myocytes cardiaques, n'entrainent pas la regenerescence du myocarde mort, et fonctionnent donc par l'intermediaire de modes d'action paracrines. L'essai SCIPIO, une collaboration entre le groupe de Harvard et le groupe de Louisville, a ete victime des fautes a Harvard. La retractation de l'article SCIPIO a ete causee exclusivement par des problemes issus des donnees produites a Harvard, pas de celles produites a Louisville. Dans l'acte de retractation, les editeurs du Lancet ont declare que bien qu'ils n'aient aucune reserve quant au travail clinique effectue a Louisville ou des preparations provenant du laboratoire d'Anversa ont ete utilisees de bonne foi, le manque de fiabilite du travail de laboratoire a Harvard implique qu'ils retractent cet article. Nous devons prendre bien garde de ne pas disqualifier tout le travail sur les CPC en raison de fautes commises dans un seul laboratoire. Une synthese non biaisee de la litterature appuie le potentiel therapeutique des CPC pour l'insuffisance cardiaque aux stades precliniques. [Traduit par la Redaction] Mots-cles : cellules progenitrices cardiaques, c-kit, cellules souches, therapie cellulaire, cardiomyopathie ischemique chronique, infarctus du myocarde., Introduction The most tragic development in the field of cell-based therapies for heart disease has been the occurrence of scientific misconduct in one high-profile laboratory at Harvard Medical School (Oransky [...]

The present study sought to determine whether the combination of late preconditioning (PC) with postconditioning enhances the reduction in infarct size. Chronically instrumented rats were assigned to a 45-min (subset 1) or 60-min (subset 2) coronary occlusion followed by 24 h of reperfusion. In each subset, rats received no further intervention (control) or were preconditioned 24 h before occlusion (PC), post-conditioned at the onset of reperfusion following occlusion, or preconditioned and postconditioned without (PC + postconditioning) or with the COX-2 inhibitor celecoxib (3 mg/kg ip; PC + postconditioning + celecoxib) 10 min before postconditioning. Myocardial cyclooxygenase-2 (COX-2) protein expression and COX-2 activity (assessed as myocardial levels of [PGE.sub.2]) were measured 6 min after reperfusion in an additional five groups (control, PC, postconditioning, PC + postconditioning, and PC + postconditioning + celecoxib) subjected to a 45-min occlusion. PC alone reduced infarct size after a 45-min occlusion but not after a 60-min occlusion. Postconditioning alone did not reduce infarct size in either setting. However, the combination of late PC and postconditioning resulted in a robust infarct-sparing effect in both settings, suggesting additive cardioprotection. Celecoxib completely abrogated the infarct-sparing effect of the combined interventions in both settings. Late PC increased COX-2 protein expression and [PGE.sub.2] content. [PGE.sub.2] content (but not COX-2 protein) was further increased by the combination of both interventions, suggesting that postconditioning increases the activity of COX-2 induced by late PC. In conclusion, the combination of late PC and postconditioning produces additive protection, likely due to a postconditioning-induced enhancement of COX-2 activity. myocardium; ischemia; infarct size; cyclooxygenase-2

Objectives: Brief episodes of ischemia and reperfusion after a lethal ischemic insult confer cardioprotection, a phenomenon termed 'ischemic postconditioning.' However, all studies reported to date have been conducted in open-chest animal models. We sought to determine whether postconditioning occurs in conscious animals and whether it protects against severe myocardial injury. Methods: Chronically instrumented rats were assigned to a 30- (Subset 1), 45- (Subset 2), or 60-min (Subset 3) coronary occlusion followed by 24 h of reperfusion. In each subset, rats received no further intervention (control), were preconditioned with 12 cycles of 2-min occlusion/2-min reperfusion immediately (early preconditioning; EPC) or 24 h (late preconditioning; LPC) before myocardial infarction, or were postconditioned with 20 cycles of 10-s occlusion/10-s reperfusion immediately after myocardial infarction (20-10 PostC). Results: With a 30-min occlusion, infarct size (54.4 [+ or -] 2.3% of risk region in control-30) was significantly reduced in EPC-30, LPC-30, and 20-10 PostC-30 groups (by 72, 70, and 47%, respectively; all P < 0.05 vs. control-30). With a 45-min occlusion, infarct size (62.2 [+ or -] 2.4% in control-45) was reduced in EPC-45 and LPC-45 groups (by 47 and 41%, respectively; all P < 0.05 vs. control-45) but not in the 20-10 PostC-45 group [55.4 [+ or -] 2.3%, P = not significant (NS) vs. control-45]. With a 60-min occlusion, infarct size (72.7 [+ or -] 2.2% in control-60) was reduced in the EPC-60 (by 20%, P < 0.05) but not in the LPC-60 (63.6 [+ or -] 2.5%, P = NS) or in the 20-20 PostC group (71.5 [+ or -] 3.4%, P = NS). Conclusions: Both early and late ischemic preconditioning as well as ischemic postconditioning confer protection in conscious rats; however, unlike early preconditioning, postconditioning protects only against coronary occlusions myocardium; ischemia; infarct size; preconditioning

Tang, Xian-Liang, Yu-Ting Xuan, Yanqing Zhu, Gregg Shirk, and Roberto Bolli. Nicorandil induces late preconditioning against myocardial infarction in conscious rabbits. Am J Physiol Heart Circ Physiol 286: H1273-H1280, 2004. First published December 18, 2003; 10.1152/ajpheart.0l055.2003.--Nicorandil has been shown to induce an infarct-limiting effect similar to that induced by the early phase of ischemic preconditioning (PC). The goals of this study were to determine whether nicorandil induces a delayed cardioprotection that is analogous to the late phase of ischemic PC and, if so, whether nicorandil-induced late PC is associated with upregulation of cardioprotective proteins. Chronically instrumented, conscious rabbits received vehicle (intravenous normal saline; control group, n = 10), nicorandil (100 [micro]g/kg bolus + 30 [micro]g.[kg.sup.-1].[min.sup.-1] iv for 60 min; nicorandil group, n = 10), or ischemic PC (6 cycles of 4-min coronary occlusion/4-min reperfusion; PC group, n = 8). Twenty-four hours later, rabbits underwent a 30-min coronary occlusion, followed by 3 days of reperfusion. Myocardial infarct size was significantly reduced in rabbits pretreated with nicorandil (27.5 [+ or -] 5.3% of the risk region) or with ischemia (30.3 [+ or -] 4.2%) versus controls (59.1 [+ or -] 4.7%, P < 0.05 vs. both). Furthermore, the expression of cyclooxygenase-2 (COX-2) and Bcl-2 was significantly elevated (+38% and + 126%, respectively; P < 0.05) in myocardium of rabbits given nicorandil 24 h earlier versus controls. We conclude that nicorandil induces delayed cardioprotection against myocardial infarction similar to that afforded by the late phase of ischemic PC, possibly by upregulating COX-2 and Bcl-2. Bcl-2; cyclooxygenase-2; infarct size; ischemia-reperfusion

Previous studies indicated that activation of PKC and Src tyrosine kinases by ischemic preconditioning (PC) may participate in the activation of NF-[kappa]B. However, the molecular mechanisms underlying activation of NF-[kappa]B during ischemic PC remain unknown. In the hearts of conscious rabbits, it was found that ischemic PC (6 cycles of 4-min coronary occlusion and 4-min reperfusion) significantly induced both tyrosine (+226.9 [+ or -] 42%) and serine (+137.0 [+ or -] 36%) phosphorylation of the NF-[kappa]B inhibitory protein I[kappa]B-[alpha], concomitant with increased activation of the I[kappa]B-[alpha] kinases IKK[alpha[ (+255.0 [+ or -] 46%) and IKK[beta] (+173.1 [+ or -] 35%). Furthermore, both tyrosine and serine phosphorylation of IKB-[alpha] were blocked by pretreatment with either the nonreceptor tyrosine kinase inhibitor lavendustin-A (LD-A) or the PKC inhibitor chelerythrine (Che) (both given at doses previously shown to block ischemic PC). Interestingly, Che completely abolished PC-induced activation of IKK[alpha]/[beta], whereas LD-A had no effect. In addition, I[kappa]B-[alpha] protein level did not change during ischemic PC. Together, these data indicate that ischemic PC-induced activation of NF-[kappa]B occurs through both tyrosine and serine phosphorylation of I[kappa]B-[alpha] and is regulated by nonreceptor tyrosine kinases and PKC. ischemic preconditioning; Src tyrosine kinase; protein kinase C; signaling module; posttranslational modification

Although protein tyrosine kinases (PTKs) signaling has been implicated in the late phase of ischemic preconditioning (PC), it is unknown whether PTK signaling is necessary for the development of nitric oxide (NO) donor-induced late PC. Thus conscious rabbits underwent a sequence of six 4-min coronary occlusion (O)/4-min reperfusion (R) cycles followed by a 5-h recovery period of reperfusion for 3 consecutive days (days 1, 2, and 3). On day 0 (24 h before the 6 O/R cycles on day 1), rabbits received no treatment (control), the NO donor diethylenetriamine (DETA)/NO (DETA/NO), the PTK inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d] pyrimidine (PP2), or DETA/NO plus PP2 (DETA/NO + PP2). In control rabbits (n = 6), the six O/R cycles on day I resulted in delayed functional recovery, indicating severe myocardial stunning. In rabbits pretreated with DETA/NO (n = 5) on day 1, myocardial stunning caused by the six O/R cycles on day I was markedly attenuated, with a significant reduction (~60%) in the total deficit of wall thickening (WTh) compared with controls, indicating that DETA/NO induced a late PC effect against stunning. However, in rabbits pretreated with DETA/NO + PP2 (n = 5), the total deficit of WTh was significantly greater than that in rabbits treated with DETA/NO alone and was similar to that in controls, indicating that PP2 prevented the development of DETA/NO-induced late PC. In rabbits pretreated with PP2 on day 0 (n = 4), the total deficit of WTh was similar to that in controls, indicating that PP2 does not affect myocardial stunning in itself. We conclude that a PTK-dependent signaling mechanism is necessary for the development of NO donor-induced late PC against myocardial stunning in conscious rabbits. diethylenetriamine/nitric oxide; pp2; ischemia-reperfusion injury; rabbit

Although activation of [delta]-opioid receptors is known to induce both early and late preconditioning (PC) against myocardial infarction, the mechanisms for this salubrious effect are unclear. Furthermore, it is unknown whether [delta]-opioid receptors can also induce late PC against myocardial stunning. By using conscious rabbits (n = 120) in this study, we found that the [delta]-opioid receptor agonist ([+ or -])-4-{([alpha]-[R.sup.*]) -[alpha]-[([2S.sup.*],[5R.sup.*])-4-allyl-2,5-dimethyl -1-piperazinyl]-3-hydroxybenzyl}-N,N-diethylbenzamide (BW-373U86) induced late PC against myocardial stunning 24 h after treatment and that this effect was abolished by the selective cyclooxygenase-2 (COX-2) inhibitors N-[2-(cyclohexyloxy)4-nitrophenyl] methanesulfonamide (NS-398) and celecoxib. This protective effect was also abrogated by the selective [[delta].sub.1]-opioid receptor antagonist 7-benzylidenenaltrexone, indicating that the [[delta].sup.1]-opioid receptor is necessary for BW-373U86-induced late PC. BW-373U86 did not induce early PC against stunning. In addition, BW-373U86 induced late PC against infarction, which was blocked by NS-398. At 24 h after BW-373U86 administration, myocardial COX-2 protein expression and PG[E.sub.2] and 6-keto-PG[F.sub.1[alpha]] levels were significantly increased. These results demonstrate that activation of [delta]-opioid receptors induces late PC against both stunning and infarction via a COX-2-dependent mechanism. ischemic-reperfused; BW-373U86; 7-benzylidenenaltrexone

Although Src protein tyrosine kinases (PTKs) have been shown to be essential in late preconditioning (PC) against myocardial stunning, their role in triggering versus mediating late PC against myocardial infarction remains unclear. Four groups of conscious rabbits were subjected to a 30-min coronary occlusion on day 2, with or without PC ischemia on day 1. Administration of the Src PTK inhibitor lavendustin A (LD-A; 1 mg/kg iv) before the PC ischemia on day 1 (group III, n = 7) failed to block the delayed protective effect against myocardial infarction 24 h later. Late PC against infarction, however, was completely abrogated when LD-A was given 24 h after the PC ischemia, prior to the 30-min occlusion on day 2 (group IV, n = 8). We conclude that, in conscious rabbits, Src PTK activity is necessary for the mediation of late PC protection against myocardial infarction on day 2, but not for the initiation of this phenomenon on day 1. Taken together with previous studies in the setting of stunning, these findings reveal heretofore unrecognized differences in the roles of Src PTKs in late PC against stunning versus late PC against infarction. lavendustin A; ischemia-reperfusion injury

Oxidant species trigger late preconditioning against myocardial stunning in conscious rabbits. Am J Physiol Heart Circ Physiol 282: H281-H291, 2002.--Conscious rabbits underwent six 4-min occlusion and 4-min reperfusion cycles for 3 consecutive days (day 1, 2, and 3); on day 1, rabbits received intravenous vehicle [preconditioning (PC)] (group I, n = 6), superoxide dismutase (SOD; group II, n = 5), catalase (group III, n = 6), or the hydroxyl radical (*OH) and peroxynitrite (ONO[O.sup.-]) scavenger N-2-mercaptopropionyl glycine (MPG [group IV], n = 6). In the PC group, the recovery of systolic wall thickening (WTh) after the sixth reperfusion was markedly improved on days 2 and 3 compared with day 1 and the total deficit of WTh was correspondingly reduced, indicating a late PC effect against myocardial stunning. Neither SOD nor catalase had any significant effect on the severity of stunning on day 1 or on the development of late PC on days 2 and 3, despite high plasma levels. In contrast, MPG markedly attenuated the severity of stunning on day 1 and prevented the development of late PC on day 2. Two additional groups of rabbits received an intracoronary infusion of vehicle (group V, n = 4) or the reactive oxygen species (ROS) generating solution [cumene hydroperoxide (CuOOH, group VI, n = 7)] on day 0, and were then subjected to the six occlusion/reperfusion cycles on days 1, 2, and 3. In group VI, infusion of CuOOH elicited a late PC effect 24 h later (on day 1). Taken together, these results demonstrate that oxidant species play an essential role in triggering the development of late PC against stunning in conscious rabbits. The fact that late PC was blocked by MPG and mimicked by CuOOH implicate ONO[O.sup.-] and/or *OH as the oxygen species responsible for the initiation of this phenomenon. In addition, the finding that exogenous ROS (CuOOH) reproduced the phenotype of late PC indicates that ROS are not only necessary but also sufficient to trigger this defensive adaptation of the heart to stress. catalase; cumene hydroperoxide; myocardial ischemia-reperfusion; N-2-mercaptopropionyl glycine; reactive oxygen species; superoxide dismutase

An experiment was conducted to determine whether early and late phases of ischemic preconditioning exist in the mouse. If so, the study then aimed to compare their relative potencies and to establish whether the magnitude of the preconditioning protection could accurately be assessed with reperfusion intervals as short as four hours or whether survival surgery was required. It was also desired to develop reliable murine models of early and late preconditioning in which basic physiological variables are carefully controlled and kept within normal values. Results are discussed.

A study was conducted to test the hypothesis that late preconditioning (PC) against myocardial stunning is related to an increase in endogenous antioxidant defenses. There was no alteration in antioxidant defenses when a marked protection against stunning was present. The results suggest that, in conscious pigs, late PC against stunning is not mediated by increased endogenous antioxidant defenses.

A study was conducted to determine the functions of adenosine A1 receptors in late preconditioning (PC) against myocardial stunning in a conscious rabbit model. The rabbits were subjected to coronary artery occlusion with interspersed reperfusion. Systolic wall thickening of artery indicated late preconditioning to stunning. Results also showed that adenosine A1 receptors contribute to early and late PC against infarction and stunning. The conscious rabbit model proved to be useful in stunning studies due to its inexpensiveness.