Copyright © 2007, European Society of Cardiology
The myocardial no-reflow phenomenon: Role of 
PKC
aDivision of Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada M5G 1X8
bDepartment of Laboratory Medicine, Division of Pathology, Hospital for Sick Children, Toronto, ON, Canada
cDepartments of Laboratory Medicine and Pathobiology, Physiology and Surgery, University of Toronto, ON, Canada
* Corresponding author. Division of Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada M5G 1X8. Tel.: +1 416 813 2095; fax: +1 416 813 5965. Email address: gregory.wilson{at}sickkids.ca
Received 4 January 2007; accepted 8 January 2007
See article by Ikeno et al. [1] (pages 699–709) in this issue.
In the paper "Impaired perfusion after myocardial infarction is due to reperfusion-induced 
PKC-mediated myocardial damage" by Ikeno et al. [1], Dr. Daria Mochly-Rosen's group at Stanford University have demonstrated a novel molecular interventional strategy, targeted inhibition of the isoform of protein kinase C (PKC), to treat the post-ischemic no-reflow phenomenon in the myocardium using both mouse and pig models. This they achieved by either expressing a PKC-specific translocation inhibitor protein fragment, V1-1, only in cardiomyocytes in a transgenic crystalloid perfused mouse ex-vivo acute global myocardial ischemia (30 min)/reperfusion (120 min) model or through intra-coronary delivery of the TAT-conjugated synthetic peptide V1-1 during the last minute of ischemia in a porcine in-vivo regional (LAD) myocardial ischemia (30 min)/reperfusion (30 min, 24 h, 6 and 12 days) model.
Their mouse experiments showed a roughly 70% reduction in both infarction and coronary vascular resistance 5 min after reflow compared to wild-type hearts. Direct coronary delivery of the V1-1 peptide in wild-type hearts provided similar results and, when added to the V1-1 transgenic hearts, essentially eliminated the rise in coronary vascular resistance although no further reduction in muscle necrosis was achieved. These results suggest that PKC inhibition protects both cardiomyocytes and microvasculature, as indicated by these investigators [1].
Their pig model included the contribution of blood cells and energy demand on the in vivo ischemic heart and involved regional ischemia more closely mimicking the clinical setting of reperfusion therapy in acute myocardial infarction. The V1-1 peptide intra-coronary infusion performed as reperfusion was about to commence substantially lowered infarct size, normalized cardiac output, and greatly reduced wall motion abnormalities which persisted 12 days post-ischemia, an impressive result. Some understanding of the phenomenology involved was achieved by several interesting features of the experimental design. Coronary flow reserve was measured in response to intra-coronary adenosine and shown to decrease only slightly with V1-1 treatment and to normalize within 24 h. V1-1 reduced the no-reflow area, measured by lack of staining by the vital endothelium-specific dye thioflavin S, by nearly two thirds. Of particular interest to us was their demonstration by electron microscopy (EM) in the reperfused area at risk in the pigs that V1-1 treatment improved microvascular patency on reperfusion, specifically achieving a substantial reduction in endothelial cell swelling and capillary obstruction by erythrocytes and leukocytes. Irreversible cardiomyocyte damage (contraction bands, swollen mitochondria with disrupted cristae and amorphous matrix densities) were essentially absent in the EM biopsies from pig hearts treated with V1-1, a point to which we shall return. Furthermore, a very nice element in the experimental design was the comparison of the V1-1 treatment to the intra-coronary delivery of the 
PKC-selective activator 
RACK, during early ischemia, to mimic preconditioning. The RACK treatment reduced infarct size by about 60% but did not improve coronary flow reserve or affect hypokinesis, providing indirect support for PKC inhibition acting through vascular mechanisms and not just indirectly through effects of reduced cardiomyocyte damage on the microvasculature.
The no-reflow phenomenon recently reviewed by Reffelmann and Kloner [2] and by Galiuto and Crea [3] may be defined as "incomplete and non-uniform reperfusion at the microvascular level despite adequate re-opening of the proximal artery after a period of transient ischemia" [2]. Galiuto and Crea [3] emphasize the distinction between ‘structural no-reflow’, in which there is irreversible damage to the cells of the microvasculature, versus ‘functional no-reflow’, in which patency of intact microvessels is compromised due to vasoconstriction or microembolization. Fortunately, the microvasculature is more resistant to prolonged ischemia than cardiomyocytes, as first demonstrated by Kloner et al. [4] over 25 years ago. This provides considerable scope for reduction of mechanical compression of blood vessels by reducing cardiomyocyte swelling and contracture. However, endothelial cell swelling (short of manifest irreversible injury), leukocyte and platelet aggregation causing obstruction of the vessel lumen, and chemically mediated vasoconstriction are additional elements of this very complex phenomenon which is further complicated by the compressive forces of interstitial edema to which pathological increases in microvascular permeability contribute. A key issue in myocardial no-reflow has been the close coupling of cardiomyocyte necrosis with microvascular perfusion defects at the heart of which (pun intended) is the practical issue of whether the no-reflow phenomenon contributes to myocardial necrosis. Reffelmann and Kloner [5] were not able to achieve decoupling of myocardial necrosis from perfusion defects in rabbits with either Na+/H+ exchanger inhibition (by cariporide) or ischemic preconditioning, two of the most powerful infarct size-reducing interventions thought to work by independent mechanisms. In contrast, Ikeno et al. [1] do provide some evidence of decoupling as illustrated in part by the contrast in their results between PKC inhibition with V1-1 and PKC activation with RACK. PKC inhibition by V1-1 seems to be acting through a combination of reduced cardiomyocyte injury and reduced endothelial cell injury based on the roughly 80% reduction in cellular injury for both endothelium and cardiomyocytes as evaluated by TUNEL staining [1]. The absence of irreversible cardiomyocyte damage from the EM biopsies observed by Ikeno et al. with V1-1 treatment [1], referred to above, raises the possibility that much of the improvement in microvascular perfusion observed in this model could have arisen from reduction in the mechanical pressure exerted by cardiomyocyte swelling and contracture causing vascular compression. The reduction in endothelial cell swelling they observed on EM was impressive. The remaining question, however, is to what extent cardiomyocyte necrosis on reperfusion (which involves chemical release from the bursting of these cells) contributes to endothelial cell swelling. From the perspective of mechanistic interpretation, distinctions between apoptosis and oncosis leading to cell death are important considerations as are interpretive limitations of TUNEL staining as a sole measure for apoptosis. Ultrastructural observations, while of great interest, are derived from very small tissue samples. These and other limitations of the study, imposed primarily by technical limitations and not the resourcefulness of the authors, are well expounded upon by Dr. Mochly-Rosen and her colleagues [1].
Clinically, myocardial "no-reflow" is used both to refer to microvascular perfusion deficits after reperfusion therapy for acute myocardial infarction by thrombolysis and/or percutaneous coronary intervention as well as after mechanical interventions on narrowed coronary arteries and coronary bypass grafts without antecedent ischemia. The importance of thrombotic/atheromatous debris embolization in myocardial no-reflow in the clinical setting is increasingly recognized [6,7]. Interestingly, microembolization has been absent from almost all experimental models of myocardial no-reflow.
There is growing clinical interest in pharmacological treatment for myocardial no-reflow. Multiple drugs, primarily vasodilators including adenosine but also glycoprotein IIb/IIIa platelet receptor antagonists, are being used clinically in efforts to ameliorate no-reflow [8]. Adenosine used as an adjunct to reperfusion within 3 h of symptoms has produced an impressive 55–65% reduction in ultimate infarct size [9] although the specific contribution of no-reflow reduction to this benefit has not been quantified and adenosine does act through several mechanisms [9].
In our view, the potential advantage of using synthetic V1-1 peptide clinically for adjunctive coronary reperfusion therapy arises from the precise molecular targeting of PKC inhibition, which may act through mechanisms not achieved by available drugs and, even if through overlapping mechanisms, is likely more specific. An important question is: Just how does PKC act on both cardiomyocytes and endothelial cells – what are its end effectors? Ikeno et al. [1] point out that reperfusion increases the translocation of PKC to cardiac mitochondria where it appears to increase the generation of reactive oxygen species, among other effects. The oxyradical theme may well be what ties together the beneficial effects of PKC inhibition on both cardiomyocytes and endothelial cells, and we believe this should be explored further.
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 Top References |
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- Ikeno F., Inagaki K., Rezaee M., Mochly-Rosen D. Impaired perfusion after myocardial infarction is due to reperfusion-induced PKC-mediated myocardial damage. Cardiovasc Res (2007) 73:699–709.
[Abstract/Free Full Text] - Reffelmann T., Kloner R.A. The no-reflow phenomenon: a basic mechanism of myocardial ischemia and reperfusion. Basic Res Cardiol (2006) 101:359–372.[CrossRef][Web of Science][Medline]
- Galiuto L., Crea F. No-reflow: a heterogeneous clinical phenomenon with multiple therapeutic strategies. Curr Pharm Des (2006) 12:3807–3815.[CrossRef][Web of Science][Medline]
- Kloner R.A., Rude R.E., Carlson N., Maroko P.R., DeBoer L.W., Braunwald E. Ultrastructural evidence of microvascular damage and myocardial cell injury after coronary artery occlusion: which comes first? Circulation (1980) 62:945–952.
[Abstract/Free Full Text] - Reffelmann T., Kloner R.A. Is microvascular protection by cariporide and ischemic preconditioning causally linked to myocardial salvage? Am J Physiol Heart Circ Physiol (2003) 284:H1134–H1141.
[Abstract/Free Full Text] - Hara M., Saikawa T., Tsunematsu Y., Sakata T., Yoshimatsu H. Predicting no-reflow based on angiographic features of lesions in patients with acute myocardial infarction. J Atheroscler Thromb (2005) 12:315–321.[Medline]
- Iijima R., Shinji H., Ikeda N., Itaya H., Makino K., Funatsu A., et al. Comparison of coronary arterial finding by intravascular ultrasound in patients with "transient no-reflow" versus "reflow" during percutaneous coronary intervention in acute coronary syndrome. Am J Cardiol (2006) 97:29–33.[CrossRef][Web of Science][Medline]
- Porto I., Ashar V., Mitchell A.R. Pharmacological management of no reflow during percutaneous coronary intervention. Curr Vasc Pharmacol (2006) 4:95–100.[CrossRef][Medline]
- Forman M., Stone G., Jackson E. Role of adenosine as adjuntive therapy in acute myocardial infarction. Cardiovasc Drug Rev (2006) 24:116–147.[CrossRef][Web of Science][Medline]
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