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Rationale:Treatment of heart failure is limited due to the limited regenerative capacity of cardiomyocytes. Our group has shown that ectopic expression of Cdk1/CyclinB1 and Cdk4/CyclinD1 (named 4F) via adenovirus vector promotes cardiomyocyte proliferation in 20% of infected cardiomyocytes in vitroand in vivoand improves cardiac function after myocardial infarction (MI).Objective:To introduce 4F specifically and transiently into cardiomyocytes to induce selective cardiomyocyte regeneration and limit any tumorigenic potential in other organs.Methods and Results:A polycistronic non-integrating lentivirus encoding 4F, each driven by a TNNT2 promoter (TNNT2-4F-NIL), was generated to induce transient and specific expression of 4F cardiomyocytes. TNNT2-4F-NIL promotes proliferation of 15% of cardiomyocytes in vivo after MI as assessed in lineage-tracing mice (α-MHC-Cre-MADM). Rats and pigs were subjected to coronary occlusion/reperfusion to induce MI. One week later, animals were injected (intramyocardially) with either TNNT2-4F-NIL or control-NIL. Four weeks after injection, TNNT2-4F-NIL treated rats and pigs showed a significant improvement in left ventricular ejection fraction (EF) compared to controls (TNNT2-4F-NIL rats 54.3±1.78% vs control-NIL 42.97±2.60%; n=9/group, p=0.003, TNNT2-4F-NIL pigs 37±2.3% vs control-NIL 28.4±1.2%; n=6/group, p=0.01). Histological analyses showed a 25-30% reduction in scar size in both rats and pigs treated with TNNT2-4F-NIL vs. control-NIL (p<0.05). To assess the long-term efficacy and safety of TNNT2-4F-NIL, a 4-months follow-up experiment after MI and virus treatment was performed during which cardiac function was assessed monthly. TNNT2-4F-NIL treated rats showed sustained improvement in cardiac function (EF in TNNT2-4F-NIL 48.4±3% vs in control-NIL 33.6±3%; n=6/group, p=0.008). There was no obvious development of arrhythmias and no evidence of oncogenicity or toxicity in other organs in TNNT2-4F-NIL treated rats or in the control group.Conclusion:This study provides a novel approach to treat heart failure, with the use of transient and cardiomyocyte-specific gene therapy that promotes long-term functional improvement after a single treatment with no obvious adverse effect.

Activation of protein kinase C (PKC) ε by nitric oxide (NO) has been implicated in the development of cardioprotection. However, the cellular mechanisms underlying the activation of PKCε by NO remain largely unknown. Nitration of protein tyrosine residues has been shown to alter functions of a variety of proteins, and NO-derived peroxynitrite is known as a strong nitrating agent. In this investigation, we demonstrate that NO donors promote translocation and activation of PKCε in an NO- and peroxynitrite-dependent fashion. NO induces peroxynitrite-mediated tyrosine nitration of PKCε in rabbit cardiomyocytes in vitro, and nitrotyrosine residues were also detected on PKCε in vivoin the rabbit myocardium preconditioned with NO donors. Furthermore, coimmunoprecipitation of PKCε and its receptor for activatedCkinase, RACK2, illustrated a peroxynitrite-dependent increase in PKCε-RACK2 interactions in NO donor-treated cardiomyocytes. Moreover, using an enzyme-linked immunosorbent assay-based protein-protein interaction assay, PKCε proteins treated with the peroxynitrite donor SIN-1 exhibited enhanced binding to RACK2 in an acellular environment. Our data demonstrate that post-translational modification of PKCε by NO donors, namely nitration of PKCε, facilitates its interaction with RACK2 and promotes translocation and activation of PKCε. These findings offer a plausible novel mechanism by which NO activates the PKC signaling pathway.

Recent studies implicate iNOS as the mediator of the late phase of ischemic preconditioning (PC). However, it is unknown whether induction of iNOS activity is mediated by transcriptional, post-transcriptional, translational, or post-translational mechanisms. To address this issue, we isolated and sequenced a partial iNOS cDNA expressed in preconditioned rabbit myocardium. Using a rabbit-specific probe generated from this sequence, we measured the steady state levels of the iNOS transcript after ischemic PC [six cycles of 4-min occlusion/4-min reperfusion (O/R)]. Three hours after ischemic PC, the iNOS mRNA levels in the ischemic/reperfused region were increased approximately three-fold relative to samples from the non-ischemic region and from control rabbits. This increase in mRNA levels was completely abolished by pretreatment with the NOS inhibitor Nω-nitro- l-arginine. Conversely, administration of the NO donor nitroglycerin induced an increase in iNOS mRNA levels similar to that induced by ischemic PC. We conclude that in the conscious rabbit, ischemic PC induces an increase in iNOS mRNA levels, and that this induction is triggered by increased generation of NO during the PC stimulus. These results provide direct evidence that upregulation of iNOS is a natural response of the heart to a brief ischemic stress and that NO itself, in the absence of ischemia, upregulates myocardial iNOS transcript levels, a finding that may have implications for nitrate therapy. This previously unrecognized NO-dependent upregulation of iNOS mRNA is likely to play an important role in the development of late PC as well as in many other pathophysiological conditions in which NO is implicated.

Recent studies suggest that calcium channel blockers attenuate reversible post-ischemic myocardial dysfunction (myocardial “stunning”)in vivo. This beneficial effect, however, has been shown either in open-chest preparations, which are subject to the confounding influence of many unphysiological conditions, or in models in which treatment caused significant hemodynamic alterations. Furthermore, all of the studies have been conducted in the dog, and almost all of them have examined the effect of calcium antagonists after a single ischemic episode. The goal of the present investigation was to assess the effect of nisoldipine in a conscious pig model of repetitive ischemia, and to determine whether the drug exerts direct cardioprotection independent of hemodynamic changes. A total of 33 conscious pigs were used. Pigs underwent a sequence of 10 2-min coronary occlusions, each separated by 2 min of reperfusion, and were randomly assigned to a treated group (n=11), in which nisoldipine was infused at a rate of 0.5μg/kg/min from 15 min before the first coronary occlusion till 30 min after the last reperfusion, and a control group (n=12), which received vehicle. Results showed that there were no significant differences between the two groups with respect to ischemic bed size or hemodynamic variables throughout the experiment. Collateral blood flow to the ischemic regions was virtually nil in both groups. During the sequence of coronary occlusions, systolic thickening fraction in the ischemic region decreased similarly in the two groups. After the 10th reperfusion, however, the recovery of wall thickening was markedly enhanced in treated compared to control pigs, with the differences being statistically significant at 5, 15, and 30 min and 1, 3, 4 and 5 h. The total deficit of wall thickening after the 10th reperfusion (an integrative assessment of post-ischemic dysfunction) was 51% less in the treated compared with the control group (P<0.001). This study demonstrates that nisoldipine markedly attenuates myocardial stunning after multiple ischemic episodes in conscious pigs, the improvement is evident immediately after the end of the ischemic episodes and is sustained throughout the recovery phase. This beneficial effect is independent of any favourable hemodynamic changes, and therefore indicates a direct cardioprotective action of nisoldipine.

A previous study has demonstrated that the hydrophilic (α-tocopherol analogue, MDL 74, 405, attenuates postischemic myocardial dysfunction ("stunning") in dogs. The present study was undertaken to determine directly whether the salutary effect of this drug on myocardial stunning results from inhibition of the generation of oxygen-derived free radicals. Open-chest dogs undergoing a 15-min coronary artery occlusion and 3 h of reperfusion received an intravenous infusion of either saline (controls, n = 7) or MDL 74, 405 (n = 6) starting 30 min before coronary occlusion and ending 60 min after reflow at a dose of 0.3 mg/kg/h. To measure free radical production, all dogs received an intravenous infusion of the spin trap α-phenyl N-tert-butyl nitrone (PBN) and local coronary venous plasma was analyzed by electron paramagnetic resonance (EPR). In control dogs, the myocardial production of PBN adducts exhibited an initial burst immediately after the onset of reflow and remained elevated until 10 min after reperfusion. Dogs treated with MDL 74, 405 demonstrated a marked decrease in PBN adduct production. This effect of MDL 74, 405 could not be attributed to nonspecific factors such as differences in ischemic zone size, collateral flow, arterial pressure, heart rate, coronary flow or other hemodynamic variables. These results demonstrate that the hydrophilic vitamin E analogue, MDL 74, 405, inhibits free radical generation after myocardial ischemia-reperfusion in vivo. This finding provides direct evidence that the salutary effects of MDL 74, 405 on myocardial stunning are due to attenuation of oxidative stress.

Rationale:Heart failure kills 1 in 10 people over the age of 65 in the USA. Recently, we identified a combination of four cell cycle regulators, cyclin-dependent kinase 1 (CDK1), CDK4, cyclin B1, and cyclin D1 (4F), that induces stable cytokinesis in adult cardiomyocytes to replace their loss following myocardial infarction (MI). Currently, our approach is the most robust method to induce cardiomyocyte proliferation; however, clinical applicability is limited by concerns for tumorigenic potential in other organs.Objective:To demonstrate that transient and cardiomyocyte-specific expression of 4F is sufficient to induce cardiomyocyte proliferation, thus avoiding potential adverse effects on other tissues.Methods and Results:Non-integrating lentivirus encoding TNNT2 promoter driving expression of the 4F (TNNT-4F-NIL) was used to induce transient and cardiomyocyte specific expression of the 4F in human-iPS-derived-cardiomyocytes (hiPS-CMs) and not in other tested cell types (fibroblasts, endothelial, skeletal muscle, and kidney cell lines). TNNT-4F-NIL induced robust proliferation markers in hiPS-CMs as indicated by EDU and PHH3 expression (28.24?3.47%, 22.04?0.90, respectively). Using our novel cytokinesis reporter (AuroraKB promoter-GFP based reporter), 25.42?2.32% of hiPS-CMs overexpressing TNNT-4F-NIL showed GFP expression vs. negligible reporter expression in control hiPS-CMs. In vivo, rats were subjected to a 2 hours coronary occlusion/reperfusion; 1 week later, TNNT-4F-NIL or TNNT-GFP-NIL control virus was injected intramyocardially into 5 sites around the infarct. 4 weeks later, cardiac function was assessed by echocardiography and hearts were collected for histological analysis. Rats injected with TNNT-4F-NIL showed a significant improvement in LV function compared to controls (ejection fraction in TNNT-4F-NIL: 54.3?1.78%, control: 42.97?2.60%; n=9/group, p=0.003). Histological analyses (trichrome stain) showed a 30% reduction (n=9/group, p=0.02) in scar size in rats that received TNNT-4F-NIL vs. control group.Conclusions:TNNT-4F-NIL improves cardiac function and structure after MI. Due to its safety and effectiveness, it has a high potential for clinical translation into heart failure gene therapy.

Objective: Postischaemic myocardial dysfunction (“stunning”) is caused in part by the generation of reactive oxygen species resulting in oxidant stress. The aim of this study was to test the hypothesis that the systemic administration of MDL 74,405, a hydrophilic cardioselective a tocopherol analogue, is beneficial in attenuating myocardial stunning. Methods: Open chest dogs undergoing a 15 min coronary artery occlusion and 4 h of reperfusion received an intravenous infusion of either saline (controls, n = 9) or MDL 74,405 (n = 8) at a dose of 0.3 mg·kg−1·h−1 starting 30 min before coronary occlusion and ending 60 min after reflow. Results: Regional myocardial function (assessed as systolic wall thickening) was similar in control and treated groups at baseline and during ischaemia. Following reperfusion, however, the dogs treated with MDL 74,405 had significant improvement in the recovery of function, which was evident 2 h after restoration of flow and was sustained throughout the rest of the reperfusion phase. Analysis of covariance showed that the differences in wall thickening after reperfusion between the two groups were independent of collateral flow during occlusion. Furthermore, the enhanced recovery effected by MDL 74,405 could not be attributed to non-specific factors such as coronary flow after reperfusion, arterial pressure, heart rate, or other haemodynamic variables. Measurements of MDL 74,405 showed that the myocardial content of the antioxidant at 4 h of reperfusion was approximately 30 times greater than the plasma concentration at 1 h of reperfusion, suggesting preferential cardiac accumulation of this drug. Conclusions: This study shows (1) that systemic administration of the a tocopherol analogue MDL 74,405 in the setting of myocardial ischaemia and reperfusion does not result in adverse cardiovascular effects, at least in the first few hours after injection; (2) that the drug accumulates in myocardial tissue at concentrations much higher than in the plasma; (3) that intravenous infusion of MDL 74,405 produces an attenuation of myocardial stunning comparable to that previously observed with intracoronary administration of other antioxidants; and (4) that this beneficial effect is independent of non-specific actions on haemodynamic variables or coronary flow. The results suggest that the systemic administration of hydrophilic, cardioselective a tocopherol analogues represents an effective therapeutic approach to the alleviation of postischaemic dysfunction. Cardiovascular Research 1994;28:235-241