Gene Therapy for Diabetic Cardiomyopathy: A New Approach for a Difficult Problem
Authors: Georges E. Haddad
Journal: Molecular Therapy (2006)

Diabetic cardiomyopathy is associated with both insulin-dependent and non-insulin-dependent diabetes mellitus and is characterized by both systolic and diastolic dysfunction [1Beckman J.A. Creager M.A. Libby P. Diabetes and atherosclerosis: epidemiology, pathophysiology, and management.JAMA. 2002; 287: 2570-2581Crossref PubMed Google Scholar, 2Diamant M. et al.Diastolic dysfunction is associated with altered myocardial metabolism in asymptomatic normotensive patients with well-controlled type 2 diabetes mellitus.J. Am. Coll. Cardiol. 2003; 42: 328-335Crossref PubMed Scopus (318) Google Scholar, 3Diamant M. et al.Diabetic cardiomyopathy in uncomplicated type 2 diabetes is associated with the metabolic syndrome and systemic inflammation.Diabetologia. 2005; 48: 1669-1670Crossref PubMed Scopus (40) Google Scholar, 4Fang Z.Y. et al.Patients with early diabetic heart disease demonstrate a normal myocardial response to dobutamine.J. Am. Coll. Cardiol. 2003; 42: 446-453Crossref PubMed Scopus (98) Google Scholar]. In clinical practice, it is often difficult to ascertain whether the influence of diabetes on left ventricular function results from direct metabolic effects on the myocardium or whether it is secondary to concomitant coronary artery disease and hypertension, which are common comorbid conditions associated with diabetes [1Beckman J.A. Creager M.A. Libby P. Diabetes and atherosclerosis: epidemiology, pathophysiology, and management.JAMA. 2002; 287: 2570-2581Crossref PubMed Google Scholar, 2Diamant M. et al.Diastolic dysfunction is associated with altered myocardial metabolism in asymptomatic normotensive patients with well-controlled type 2 diabetes mellitus.J. Am. Coll. Cardiol. 2003; 42: 328-335Crossref PubMed Scopus (318) Google Scholar, 3Diamant M. et al.Diabetic cardiomyopathy in uncomplicated type 2 diabetes is associated with the metabolic syndrome and systemic inflammation.Diabetologia. 2005; 48: 1669-1670Crossref PubMed Scopus (40) Google Scholar]. A number of animal models of diabetes have proven useful for the study of the pathophysiological mechanisms that result in diabetic cardiomyopathy [5Mahgoub M.A. Abd-Elfattah A.S. Diabetes mellitus and cardiac function.Mol. Cell Biochem. 1998; 180: 59-64Crossref PubMed Scopus (115) Google Scholar]. In this issue of Molecular Therapy, Sakata and colleagues make use of a model characterized by extensive cardiac dysfunction and show that they can reverse not only the contractile dysfunction but also mechano-energetic abnormalities by specifically targeting the sarcoplasmic reticulum calcium ATPase pump (SERCA2a) [6Sakata S. et al.Mechanical and metabolic rescue in a type II diabetes model of cardiomyopathy by targeted gene transfer.Mol. Ther. 2006; 13 (this issue.)Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar]. The molecular mechanisms underlying the pathophysiology of diabetes-induced cardiomyopathy have been studied extensively in animal models [7Depre C. et al.Streptozotocin-induced changes in cardiac gene expression in the absence of severe contractile dysfunction.J. Mol. Cell. Cardiol. 2000; 32: 985-996Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 8Razeghi P. et al.Metabolic gene expression in fetal and failing human heart.Circulation. 2001; 104: 2923-2931Crossref PubMed Google Scholar, 9Taegtmeyer H. McNulty P. Young M.E. Adaptation and maladaptation of the heart in diabetes: Part I: general concepts.Circulation. 2002; 105: 1727-1733Crossref PubMed Scopus (469) Google Scholar, 10Young M.E. et al.Impaired long-chain fatty acid oxidation and contractile dysfunction in the obese Zucker rat heart.Diabetes. 2002; 51: 2587-2595Crossref PubMed Google Scholar]. These models can be produced by exposure of animals to pharmaceuticals such as streptozotocin, which leads to a condition that is similar to type I diabetes [7Depre C. et al.Streptozotocin-induced changes in cardiac gene expression in the absence of severe contractile dysfunction.J. Mol. Cell. Cardiol. 2000; 32: 985-996Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 11Rupp H. Elimban V. Dhalla N.S. Modification of myosin isozymes and SR Ca2+-pump ATPase of the diabetic rat heart by lipid-lowering interventions.Mol. Cell. Biochem. 1994; 132: 69-80Crossref PubMed Scopus (42) Google Scholar]. These models may also arise spontaneously due to genetic mutations, such as is the case with the Otsuka-Long-Evans Tokushima Fatty rats [12Abe T. et al.Left ventricular diastolic dysfunction in type 2 diabetes mellitus model rats.Am. J. Physiol. Heart Circ. Physiol. 2002; 282: H138-348Crossref PubMed Google Scholar, 13Kawano K. et al.Spontaneous long-term hyperglycemic rat with diabetic complications. Otsuka Long-Evans Tokushima Fatty (OLETF) strain.Diabetes. 1992; 41: 1422-1428Crossref PubMed Scopus (898) Google Scholar], which exhibit a condition equivalent to type II diabetes, a more common form of diabetes in humans. Sakata et al. made use of this latter model in their experiments, rendering the results quite relevant to the human condition. These animals exhibit cellular and molecular abnormalities in their heart muscle that result in functional changes that are very similar to those that occur during overt heart failure, including a recapitulation of the fetal program [7Depre C. et al.Streptozotocin-induced changes in cardiac gene expression in the absence of severe contractile dysfunction.J. Mol. Cell. Cardiol. 2000; 32: 985-996Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 8Razeghi P. et al.Metabolic gene expression in fetal and failing human heart.Circulation. 2001; 104: 2923-2931Crossref PubMed Google Scholar, 9Taegtmeyer H. McNulty P. Young M.E. Adaptation and maladaptation of the heart in diabetes: Part I: general concepts.Circulation. 2002; 105: 1727-1733Crossref PubMed Scopus (469) Google Scholar, 10Young M.E. et al.Impaired long-chain fatty acid oxidation and contractile dysfunction in the obese Zucker rat heart.Diabetes. 2002; 51: 2587-2595Crossref PubMed Google Scholar, 14Davidoff A.J. Gwathmey J.K. Pathophysiology of cardiomyopathies: Part I. Animal models and humans.Curr. Opin. Cardiol. 1994; 9: 357-368Crossref PubMed Google Scholar, 15Dutta K. et al.Cardiomyocyte dysfunction in sucrose-fed rats is associated with insulin resistance.Diabetes. 2001; 50: 1186-1192Crossref PubMed Google Scholar]. Furthermore, animal models of diabetes demonstrate significant changes in the expression of Ca2+-regulating proteins, and this change in gene expression is similar to what is observed in patients with ischemic, hypertensive, or familial cardiomyopathies [11Rupp H. Elimban V. Dhalla N.S. Modification of myosin isozymes and SR Ca2+-pump ATPase of the diabetic rat heart by lipid-lowering interventions.Mol. Cell. Biochem. 1994; 132: 69-80Crossref PubMed Scopus (42) Google Scholar, 16Dhalla N.S. et al.Pathophysiology of cardiac dysfunction in congestive heart failure.Can. J. Cardiol. 1993; 9: 873-887PubMed Google Scholar]. At the cellular level, the cardiomyopathy associated with diabetes has been characterized extensively in animal models. Abnormal excitation-contraction coupling of cardiomyocytes in streptozotocin-induced diabetes is consistent with that observed in whole hearts and multicellular muscle preparations [11Rupp H. Elimban V. Dhalla N.S. Modification of myosin isozymes and SR Ca2+-pump ATPase of the diabetic rat heart by lipid-lowering interventions.Mol. Cell. Biochem. 1994; 132: 69-80Crossref PubMed Scopus (42) Google Scholar, 16Dhalla N.S. et al.Pathophysiology of cardiac dysfunction in congestive heart failure.Can. J. Cardiol. 1993; 9: 873-887PubMed Google Scholar]. These defects in excitation-contraction coupling manifest as slowed myocyte contraction and relaxation, which resembles clinical findings in patients. Specifically, cellular changes in heart muscle from diabetic patients with cardiomyopathy include prolonged action potential (AP) duration, slowed intracellular Ca2+ transients and slowed myocyte shortening and relaxation [5Mahgoub M.A. Abd-Elfattah A.S. Diabetes mellitus and cardiac function.Mol. Cell Biochem. 1998; 180: 59-64Crossref PubMed Scopus (115) Google Scholar]. Some of the molecular changes contributing to these functional abnormalities in calcium handling have been determined in animal models and include depressed outward K+ currents (particularly Ito), Na+/Ca2+ exchange and sarcoplasmic reticulum (SR) Ca2+ uptake [7Depre C. et al.Streptozotocin-induced changes in cardiac gene expression in the absence of severe contractile dysfunction.J. Mol. Cell. Cardiol. 2000; 32: 985-996Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 11Rupp H. Elimban V. Dhalla N.S. Modification of myosin isozymes and SR Ca2+-pump ATPase of the diabetic rat heart by lipid-lowering interventions.Mol. Cell. Biochem. 1994; 132: 69-80Crossref PubMed Scopus (42) Google Scholar]. In addition, a decrease in expression of ryanodine receptors (RYR) has been described and may contribute to the contractile dysfunction. Depressed phosphorylation of phospholamban (PLB) and decreased signaling of the β-adrenergic receptor also play a role in slowed cytosolic Ca2+ clearance (see Fig. 1). Nevertheless, the exact mechanism(s) responsible for the cardiomyocyte abnormalities associated with diabetic cardiomyopathy have been difficult to ascertain, due to the presence of other metabolic changes, such as hypoinsulinemia, hyperglycemia, and dyslipidemia, which are associated with long-term (weeks to months) diabetes [12Abe T. et al.Left ventricular diastolic dysfunction in type 2 diabetes mellitus model rats.Am. J. Physiol. Heart Circ. Physiol. 2002; 282: H138-348Crossref PubMed Google Scholar, 13Kawano K. et al.Spontaneous long-term hyperglycemic rat with diabetic complications. Otsuka Long-Evans Tokushima Fatty (OLETF) strain.Diabetes. 1992; 41: 1422-1428Crossref PubMed Scopus (898) Google Scholar, 17Mizushige K. et al.Alteration in left ventricular diastolic filling and accumulation of myocardial collagen at insulin-resistant prediabetic stage of a type II diabetic rat model.Circulation. 2000; 101: 899-907Crossref PubMed Google Scholar]. As observed in most animal models of heart failure, diabetic cardiomyopathy is characterized by a decrease in expression of SERCA2a [18Date T. et al.Myocardial expression of baculoviral p35 alleviates doxorubicin-induced cardiomyopathy in rats.Hum. Gene Ther. 2003; 14: 947-957Crossref PubMed Scopus (7) Google Scholar]. As depicted in Figure 1, depolarization of the membrane by the action potential leads to the opening of voltage-gated L-type calcium (Ca2+) channels and allows a small amount of Ca2+ to enter the cell. Through a coupling mechanism between the L-type Ca2+ channel and the SR release channels (RYR), a larger amount of Ca2+ is released, leading to activation of myofilaments and contraction. During relaxation, Ca2+ is returned to the SR by SERCA2a and is then extruded extracellularly by the sarcolemmal Na+/Ca2+ exchanger (NCX). The release of Ca2+ into the myofibrillar space in turn results in cross-bridge formation and contraction. Myofilaments are organized into a regular array of thick and thin filaments, which produces the typical striated appearance of the entire myocyte. Each unit of this striated organization, known as the sarcomere, is composed of one unit of interacting thin and thick filaments. Each myosin head has an ATP-binding area in close proximity to myosin ATPase, which breaks down ATP. Two myosin heavy chain (MHC) isoforms exist in human myocardium: α- and β-MHC. α-MHC comprises 10-20% of the total in ventricular myocardium. A group of three regulatory proteins called troponin complexes carried on a long helical molecule called tropomyosin are located at intervals of 385 Angstroms along the thin filaments. The troponin complex is made up of one molecule each of troponin C, troponin I, and troponin T. The strength of the bond linking troponin I and actin varies depending on the intracellular Ca2+ level. The number of molecular targets in heart failure continues to increase with advances in the knowledge of the molecular basis of the disease. As mentioned above, the changes in calcium-cycling proteins and myofibrillar proteins are reminiscent of the changes that occur in diabetic cardiomyopathies (type I or type II) [5Mahgoub M.A. Abd-Elfattah A.S. Diabetes mellitus and cardiac function.Mol. Cell Biochem. 1998; 180: 59-64Crossref PubMed Scopus (115) Google Scholar, 7Depre C. et al.Streptozotocin-induced changes in cardiac gene expression in the absence of severe contractile dysfunction.J. Mol. Cell. Cardiol. 2000; 32: 985-996Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 9Taegtmeyer H. McNulty P. Young M.E. Adaptation and maladaptation of the heart in diabetes: Part I: general concepts.Circulation. 2002; 105: 1727-1733Crossref PubMed Scopus (469) Google Scholar, 10Young M.E. et al.Impaired long-chain fatty acid oxidation and contractile dysfunction in the obese Zucker rat heart.Diabetes. 2002; 51: 2587-2595Crossref PubMed Google Scholar, 11Rupp H. Elimban V. Dhalla N.S. Modification of myosin isozymes and SR Ca2+-pump ATPase of the diabetic rat heart by lipid-lowering interventions.Mol. Cell. Biochem. 1994; 132: 69-80Crossref PubMed Scopus (42) Google Scholar, 14Davidoff A.J. Gwathmey J.K. Pathophysiology of cardiomyopathies: Part I. Animal models and humans.Curr. Opin. Cardiol. 1994; 9: 357-368Crossref PubMed Google Scholar, 15Dutta K. et al.Cardiomyocyte dysfunction in sucrose-fed rats is associated with insulin resistance.Diabetes. 2001; 50: 1186-1192Crossref PubMed Google Scholar, 16Dhalla N.S. et al.Pathophysiology of cardiac dysfunction in congestive heart failure.Can. J. Cardiol. 1993; 9: 873-887PubMed Google Scholar]. In fact, the Otsuka-Long-Evans Tokushima Fatty rats used by Sakata et al. are also characterized by these molecular alterations [12Abe T. et al.Left ventricular diastolic dysfunction in type 2 diabetes mellitus model rats.Am. J. Physiol. Heart Circ. Physiol. 2002; 282: H138-348Crossref PubMed Google Scholar, 13Kawano K. et al.Spontaneous long-term hyperglycemic rat with diabetic complications. Otsuka Long-Evans Tokushima Fatty (OLETF) strain.Diabetes. 1992; 41: 1422-1428Crossref PubMed Scopus (898) Google Scholar, 17Mizushige K. et al.Alteration in left ventricular diastolic filling and accumulation of myocardial collagen at insulin-resistant prediabetic stage of a type II diabetic rat model.Circulation. 2000; 101: 899-907Crossref PubMed Google Scholar]. Overall, targets for molecular intervention can be divided into three categories: 1) the β-adrenergic system, 2) calcium-handling proteins, and 3) apoptotic pathways. Several gene-based approaches have been reported to restore defective β—adrenergic (βAR) signaling and to rescue the heart failure phenotype. In vitro gene transfer of β2AR and βARKct have been reported to result in augmented or rescued βAR signaling following adenovirus-mediated transfer of each gene to normal or dysfunctional rabbit cardiomyocytes, respectively [19Akhter S.A. et al.Restoration of β-adrenergic signaling in failing cardiac ventricular myocytes via adenoviral-mediated gene transfer.Proc. Natl. Acad. Sci. USA. 1997; 94: 12100-12105Crossref PubMed Scopus (168) Google Scholar, 20Maurice J.P. et al.Enhancement of cardiac function after adenoviral-mediated in vivo intracoronary β2-adrenergic receptor gene delivery.J. Clin. Invest. 1999; 104: 21-29Crossref PubMed Google Scholar, 21Rockman H.A. et al.Expression of a β-adrenergic receptor kinase 1 inhibitor prevents the development of myocardial failure in gene-targeted mice.Proc. Natl. Acad. Sci. USA. 1998; 95: 7000-7005Crossref PubMed Scopus (432) Google Scholar, 22Shah A.S. et al.In vivo ventricular gene delivery of a β-adrenergic receptor kinase inhibitor to the failing heart reverses cardiac dysfunction.Circulation. 2001; 103: 1311-1316Crossref PubMed Google Scholar, 23Jones J.M. et al.β2 adrenoceptor gene therapy ameliorates left ventricular dysfunction following cardiac surgery.Eur. J. Cardiothorac. Surg. 2004; 26: 1161-1178Crossref PubMed Scopus (12) Google Scholar]. Similarly, transcoronary delivery of adenoviruses expressing the β2AR or βARKct gene to normal rabbit heart resulted in increased contractility. [19Akhter S.A. et al.Restoration of β-adrenergic signaling in failing cardiac ventricular myocytes via adenoviral-mediated gene transfer.Proc. Natl. Acad. Sci. USA. 1997; 94: 12100-12105Crossref PubMed Scopus (168) Google Scholar, 20Maurice J.P. et al.Enhancement of cardiac function after adenoviral-mediated in vivo intracoronary β2-adrenergic receptor gene delivery.J. Clin. Invest. 1999; 104: 21-29Crossref PubMed Google Scholar, 21Rockman H.A. et al.Expression of a β-adrenergic receptor kinase 1 inhibitor prevents the development of myocardial failure in gene-targeted mice.Proc. Natl. Acad. Sci. USA. 1998; 95: 7000-7005Crossref PubMed Scopus (432) Google Scholar, 22Shah A.S. et al.In vivo ventricular gene delivery of a β-adrenergic receptor kinase inhibitor to the failing heart reverses cardiac dysfunction.Circulation. 2001; 103: 1311-1316Crossref PubMed Google Scholar, 23Jones J.M. et al.β2 adrenoceptor gene therapy ameliorates left ventricular dysfunction following cardiac surgery.Eur. J. Cardiothorac. Surg. 2004; 26: 1161-1178Crossref PubMed Scopus (12) Google Scholar]. More importantly, delivery of an adenoviral vector encoding βARKct was reported to have beneficial effects in a rabbit myocardial infarct (MI) model. In contrast to the results in small animal models, the clinical experience with pharmacological agents suggests that genetically augmenting βAR signaling may prove to be detrimental in the setting of chronic heart failure [24Stevenson L.W. Clinical use of inotropic therapy for heart failure: looking backward or forward? Part II: chronic inotropic therapy.Circulation. 2003; 108: 492-497Crossref PubMed Scopus (100) Google Scholar]. For example, ionotropic agents (which induce contractility) such as βAR agonists increase, whereas βAR antagonists decrease, mortality in patients with heart failure, suggesting that βAR desensitization occurs as a protective mechanism against the toxic effects of chronic sympathetic discharge [24Stevenson L.W. Clinical use of inotropic therapy for heart failure: looking backward or forward? Part II: chronic inotropic therapy.Circulation. 2003; 108: 492-497Crossref PubMed Scopus (100) Google Scholar]. As a result, controversy abounds in the scientific literature as to the perceived benefits and potential dangers of gene-based approaches to augment βAR. Most gene transfer approaches targeting altered calcium physiology have aimed to increase SERCA function or decrease the inhibitory effect of PLB. The net effect of either SERCA2a overexpression or PLB down-regulation is the restoration of a favorable ratio of PLB to SERCA2a, a measure that seems to be of greater physiological relevance than either change alone. In experiments targeting SERCA function, restoration of the calcium transient morphology in cardiomyocytes isolated from failing human hearts has been reported after adenovirus-mediated gene transfer of SERCA [25Matsui T. et al.Akt activation preserves cardiac function and prevents injury after transient cardiac ischemia in vivo..Circulation. 2001; 104: 330-335Crossref PubMed Google Scholar, 26del Monte F. et al.Restoration of contractile function in isolated cardiomyocytes from failing human hearts by gene transfer of SERCA2a.Circulation. 1999; 100: 2308-2311Crossref PubMed Google Scholar]. In vivo transcoronary gene transfer of the same adenoviral vector to pressure-loaded, myopathic rat hearts also led to restoration of SERCA2a levels, improved cardiac hemodynamics, and increased animal survival [26del Monte F. et al.Restoration of contractile function in isolated cardiomyocytes from failing human hearts by gene transfer of SERCA2a.Circulation. 1999; 100: 2308-2311Crossref PubMed Google Scholar, 27del Monte F. et al.Improvement in survival and cardiac metabolism following gene transfer of SERCA2a in a rat model of heart failure.Circulation. 2001; 104: 1424-1429Crossref PubMed Google Scholar]. By targeting SERCA2a, Sakata et al. show not only an improvement in contractility in these animals, but also a restoration of mechano-energetics. In fact, gene transfer of SERCA2a is being planned in a number of gene therapy trials in patients with congestive heart failure by Dr. Roger Hajjar (personal communication). Studies have also shown that decreasing the activity of PLB (the endogeneous inhibitor of SERCA2a) results in improved contractility. In fact, improved calcium handling, cardiac hemodynamics and retardation of the progression to heart failure were reported following transcoronary delivery of a recombinant adenovirus-associated virus (rAAV) encoding a dominant-negative form of PLB in a hamster model of cardiomyopathy [28Hoshijima M. et al.Chronic suppression of heart-failure progression by a pseudophosphorylated mutant of phospholamban via in vivo cardiac rAAV gene delivery.Nat. Med. 2002; 8: 864-871Crossref PubMed Scopus (324) Google Scholar]. The excitement generated by this latter study, which utilized clinically meaningful gene and vector delivery methods, has recently been tempered by the discovery of a dominant-negative mutation in PLB that causes human dilated cardiomyopathy. More recently, S100A1 was found to be an important target for the correction of calcium handling in heart failure. S100A1 is a member of a family of low molecular weight Ca2+-cycling proteins that is abundantly expressed in the myocardium [29Donato R. et al.S100: a multigenic family of calcium-modulated proteins of the EF-hand type with intracellular and extracellular functional roles.Int. J. Biochem. Cell Biol. 2001; 33: 637-668Crossref PubMed Scopus (1319) Google Scholar]. In heart failure, the expression of S100A1 is reduced, and its overexpression by gene transfer induces an increase in the activities of SERCA2a and RYR, a decrease in Nai, a decrease in NCX, and restoration of creatine phosphate levels, resulting in improved intracellular calcium handling and enhanced systolic and diastolic ventricular function in rats following cryoinfarction [30Most P. et al.Cardiac adenoviral S100A1 gene delivery rescues failing myocardium.J. Clin. Invest. 2004; 114: 1550-1563Crossref PubMed Google Scholar]. Myocardial protection by gene transfer encompasses a diverse range of strategies aimed at preventing cardiac cell injury and death [31Danel C. et al.Gene therapy for oxidant injury-related diseases: adenovirus-mediated transfer of superoxide dismutase and catalase cDNAs protects against hyperoxia but not against ischemia-reperfusion lung injury.Hum. Gene Ther. 1998; 9: 1487-1496Crossref PubMed Google Scholar, 32Li Q. et al.Gene therapy with extracellular superoxide dismutase protects conscious rabbits against myocardial infarction.Circulation. 2001; 10: 1893-1898Crossref Google Scholar, 33Yang G.Y. et al.Attenuation of ischemia-induced mouse brain injury by SAG, a redox-inducible antioxidant protein.J. Cereb. Blood Flow Metab. 2001; 21: 722-733Crossref PubMed Google Scholar]. Offending pathophysiologies cause cell death by inflammatory, immunological or oxidant stress-dependent pathways. There is growing amount of data on the role of oxidative stress in cardiovascular disease. “Stress” occurs when excess amounts of reactive oxygen species (ROS) are generated and overwhelm the endogenous antioxidant systems, resulting in cell death or dysfunction [31Danel C. et al.Gene therapy for oxidant injury-related diseases: adenovirus-mediated transfer of superoxide dismutase and catalase cDNAs protects against hyperoxia but not against ischemia-reperfusion lung injury.Hum. Gene Ther. 1998; 9: 1487-1496Crossref PubMed Google Scholar, 32Li Q. et al.Gene therapy with extracellular superoxide dismutase protects conscious rabbits against myocardial infarction.Circulation. 2001; 10: 1893-1898Crossref Google Scholar, 33Yang G.Y. et al.Attenuation of ischemia-induced mouse brain injury by SAG, a redox-inducible antioxidant protein.J. Cereb. Blood Flow Metab. 2001; 21: 722-733Crossref PubMed Google Scholar]. In general, an antioxidant effect can be created by either reducing ROS production or increasing enzymatic metabolism of these species. Models of the latter approach predominate in the literature. These models usually involve gene transfer of an antioxidant enzyme to the myocardium of an animal subjected to ischemia-reperfusion injury [31Danel C. et al.Gene therapy for oxidant injury-related diseases: adenovirus-mediated transfer of superoxide dismutase and catalase cDNAs protects against hyperoxia but not against ischemia-reperfusion lung injury.Hum. Gene Ther. 1998; 9: 1487-1496Crossref PubMed Google Scholar, 32Li Q. et al.Gene therapy with extracellular superoxide dismutase protects conscious rabbits against myocardial infarction.Circulation. 2001; 10: 1893-1898Crossref Google Scholar, 33Yang G.Y. et al.Attenuation of ischemia-induced mouse brain injury by SAG, a redox-inducible antioxidant protein.J. Cereb. Blood Flow Metab. 2001; 21: 722-733Crossref PubMed Google Scholar]. Myocardial protection after gene transfer has been reported for the enzymes manganese superoxide dismutase and heme oxygenase-1 [34Coito A.J. et al.Heme oxygenase-1 gene transfer inhibits inducible nitric oxide synthase expression and protects genetically fat Zucker rat livers from ischemia-reperfusion injury.Transplantation. 2002; 74: 96-102Crossref PubMed Google Scholar]. Apoptotic cell death of the myocardium contributes to the pathogenesis of heart failure, irrespective of etiology. As such, molecular targets within the signaling and effector pathways of apoptosis have been pursued in gene therapy approaches to treat models of heart failure, in an attempt to attenuate the burden of cell death. Support for this approach includes in vitro data demonstrating that rat cardiomyocytes can be protected from the induction of apoptosis by p53 expression or by hypoxia, following adenovirus-mediated overexpression of Bcl-2 (antiapoptotic effector) or PI-3 kinase and Akt (prosurvival signaling), respectively [25Matsui T. et al.Akt activation preserves cardiac function and prevents injury after transient cardiac ischemia in vivo..Circulation. 2001; 104: 330-335Crossref PubMed Google Scholar, 35Long X. et al.p53 and the hypoxia-induced apoptosis of cultured neonatal rat cardiac myocytes.J. Clin. Invest. 1997; 99: 2635-2643Crossref PubMed Google Scholar, 36Shimazaki K. et al.Adeno-associated virus vector-mediated bcl-2 gene transfer into post-ischemic gerbil brain in vivo: prospects for gene therapy of ischemia-induced neuronal death.Gene Ther. 2000; 7: 1244-1249Crossref PubMed Google Scholar]. In small animal models of ischemia-reperfusion injury, adenovirus-mediated gene transfer of Akt has been reported to reduce the number of apoptotic cells significantly in the region of injury, to limit infarct size and to preserve cardiac hemodynamics [25Matsui T. et al.Akt activation preserves cardiac function and prevents injury after transient cardiac ischemia in vivo..Circulation. 2001; 104: 330-335Crossref PubMed Google Scholar]. The work by Sakata et al. clearly shows that gene transfer of the calcium ATPase pump improves contractility in a model of diabetes induced cardiomyopathy. The important finding in this study is that the authors find an enhancement in energetic parameters. Pharmacological agents used in the clinic that increase contractility in heart failure by increasing cAMP, induce worsening energetic imbalance, arrhythmias and worsen survival. In fact, these types of agents have been shown to worsen morbidity and mortality in patients with heart failure. Therefore, targeting calcium cycling specifically in heart failure may provide great advantage over standard inotropic agents. This study raises a number of questions about the applicability of gene-therapy strategies in humans. First generation adenoviruses were used to induce the expression of SERCA2a, and the time points studied following gene transfer were relatively early. Further studies using AAV- or lentivirus-based vectors that examine the effects of long-term expression would be useful. rAAV vectors have a number of clinically favorable attributes, such as the lack of pathogenicity and vector-related cytotoxicity, minimal immunogenecity, and the capacity for stable long-term transgene expression through genomic integration and/or stable episome maintenance [37Du L. et al.Differential myocardial gene delivery by recombinant serotype-specific adeno-associated viral vectors.Mol Ther. 2004; 10: 604-608Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 38Negishi A. et al.Analysis of the interaction between adeno-associated virus and heparan sulfate using atomic force microscopy.Glycobiology. 2004; 14: 969-977Crossref PubMed Scopus (29) Google Scholar, 39Rabinowitz J.E. et al.Cross-dressing the virion: the transcapsidation of adeno-associated virus serotypes functionally defines subgroups.J. Virol. 2004; 78: 4421-4432Crossref PubMed Scopus (139) Google Scholar]. Different AAV vectors show different tropisms for specific organs. For example, vectors based on AAV1, AAV6, and AAV8 show a high degree of expression in skeletal muscle, and to a certain degree in cardiac muscle [40Wang Z. et al.Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart.Nat. Biotechnol. 2005; 23: 321-328Crossref PubMed Scopus (500) Google Scholar]. These vectors are tropic for striated muscle and can confer stable long-term transgene expression in skeletal and cardiac muscle in immunocompetent hosts. AAV8 has been found to be highly efficient in crossing the blood vessel barrier and in transducing skeletal and cardiac muscle [40Wang Z. et al.Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart.Nat. Biotechnol. 2005; 23: 321-328Crossref PubMed Scopus (500) Google Scholar]. Using AAV8, investigators targeted a genetic strain of Syrian hamsters with a large deletion in the β-sarcoglycan (SG) gene (TO-2 strain). This genetic strain is characterized by skeletal muscle myopathy, and severe cardiac dilation and cardiomyopathy, which primarily contributes to the very high mortality in these animals. In fact, Zhu et al. reported that a single injection of an AAV8 vector carrying the human β-SG gene enhanced contractile function in the hearts of these diseased hamsters, restored exercise capacity and survival to normal, and improved skeletal muscle function [41Zhu T. et al.Sustained whole-body functional rescue in congestive heart failure and muscular dystrophy hamsters by systemic gene transfer.Circulation. 2005; 112: 2650-2659Crossref PubMed Scopus (79) Google Scholar]. More recently, a AAV9-based vector was shown to be highly tropic for the heart, exhibiting a level of cardiac expression that was much greater than that observed using a vector based on AAV8 [42Sarkar R. et al.Long-Term Efficacy of Adeno-associated Virus Serotypes 8 and 9 in Hemophilia A Dogs and Mice.Hum. Gene Ther. 2006; (In the press.)Crossref PubMed Scopus (85) Google Scholar]. Lentiviral and retroviral vectors can transduce target cells by genomic integration. In contrast to retroviral vectors, lentiviral vectors are capable of transducing mitotically quiescent cells, a property that broadens the range of target cells, particularly within the cardiovascular system. The most commonly used lentiviral vector system is based on the human immunodeficiency virus type 1 (HIV-1). These vectors have been used to transduce cardiac tissue in vivo. In addition, large animal studies are warranted to confirm the results of Sakata et al., since the response to enhanced calcium cycling may vary between rodents and larger mammals. The contribution of SERCA2a and NCX to the lowering of cytosolic Ca2+ levels varies between species. In humans, approximately 75% of the Ca2+ is removed by SERCA2a and approximately 25% is removed by NCX, whereas in rodents close to 95% is removed by SERCA2a [43Bers D.M. et al.Cardiac excitation-contraction coupling.Nature. 2002; 415: 198-205Crossref PubMed Scopus (3297) Google Scholar]. Furthermore, the effects of SERCA2a overexpression were not studied for arrhythmogenic potential. Finally, in chronic diabetes, there are multiple changes that occur to proteins, such as glycation, that cannot be easily corrected by gene transfer. Overall, this is a significant study showing that molecular targeting can increase contractility in the failing heart by enhancing calcium cycling. This type of strategy, once tested with more clinically relevant vectors and employing cardiac-specific promoters in large animals may represent a new treatment for the failing heart, which will ultimately lead to clinical trials in gene therapy for congestive heart failure.