Effects of cyanamide, an aldehyde dehydrogenase type 2 inhibitor, on glyceryl trinitrate- and isosorbide dinitrate-induced vasodilation in rabbit excised aorta and in anesthetized whole animal.

The contribution of aldehyde dehydrogenase type 2 (ALDH2) to bioactivation of glyceryl trinitrate (GTN) and isosorbide dinitrate (ISDN) was systematically examined in excised rabbit aorta and anesthetized whole animal with cyanamide, an ALDH2 inhibitor. In excised aortic preparation, the degree of inhibition by cyanamide in GTN-induced vasorelaxation (concentration ratio, calculated as EC(50) in the presence of cyanamide/EC(50) in the absence of cyanamide; 5.61) was twice that in ISDN-induced relaxation (2.78). However, the degree of inhibition by cyanamide, as assessed by the dose ratio (as described above, but calculated with doses) in anesthetized rabbits was 2.29 in GTN-induced hypotension (assessed by area under the curve (AUC) of 50 mmHg·min) and 7.68 in ISDN-induced hypotension. Thus, the inhibitor was 3 times more potent in ISDN-induced hypotension, a finding in conflict with to that obtained in excised aortic preparation. The rate of increase in plasma nitrite (NO(2)(-)) concentration at certain hypotensive effect (50 mmHg·min of AUC) in the presence and absence of cyanamide (ΔNO(2)(-) ratio) was larger in ISDN-induced hypotension (15.01) than in GTN-induced hypotension (3.28). These results indicate that the bioactivation pathway(s) of GTN is ALDH2-dependent in aortic smooth muscle, while ADLH2-independent mechanism(s) largely take place in the whole body. In contrast, the activation mechanism(s) of ISDN is largely ALDH2-dependent in both aortic smooth muscle and whole body. Plasma NO(2)(-) may be derived from pathways other than the cyanamide-sensitive metabolic route.

Possible involvement of hypothalamic nucleobindin-2 in hyperphagic feeding in Tsumura Suzuki obese diabetes mice.

The aim of this study was to clarify the hypothalamic neuropeptides that are associated with hyperphagic feeding in Tsumura Suzuki Obese Diabetes (TSOD) mice, a model of type 2 diabetes with polygenic abnormalities. TSOD mice showed an increase in body weight and hyperleptinemia from 1 month of age and hyperphagic feeding, hyperglycemia, hyperlipidemia and hyperinsulinemia from 3 to 12 months of age compared with age-matched non-diabetic control Tsumura Suzuki Non Obesity (TSNO) mice. The mRNA level of nucleobindin-2 (NUCB2), the precursor of the anorexigenic neuropeptide nesfatin-1, was significantly decreased in the hypothalamus of TSOD mice compared with that in TSNO mice from 3 to 12 months of age. The protein level of NUCB2 was significantly decreased in the hypothalamus of TSOD mice compared with that in TSNO mice at 3 months of age. The mRNA levels of galanin, melanin-concentrating hormone, neuropeptide Y, and pro-opiomelanocortin were significantly changed in the hypothalamus in TSOD mice at several time points. Another model of type 2 diabetes, db/db mice, which is a mutant mouse that lacks a functional leptin receptor, showed hyperphagic feeding but no change in hypothalamic NUCB2 mRNA compared with non-diabetic control db/+ mice. The results suggest that the disrupted control of hypothalamic NUCB2-mediated signaling may contribute to hyperphagic feeding in TSOD mice. In addition, the mechanism for the development of hyperphagic feeding in TSOD mice is different than that in db/db mice.

Role of plasma S-nitrosothiols in regulation of blood pressure in anesthetized rabbits with special references to hypotensive effects of acetylcholine and nitrovasodilators.

The regulatory role of plasma nitrosothiols (R-SNOs) under steady-state conditions and their possible contribution to pharmacological vasodilation were systematically examined in anesthetized rabbits. Nitrosocystein (Cys-NO), S-nitrosoglutathione (G-SNO), and S-nitrosoalbumin (Alb-SNO) were determined by HPLC-Saville's method with respective sensitivities of 1, 1, and 5 nM. These R-SNOs were not detected under steady-state conditions even in the presence of N-ethylmaleimide, a thiol protective agent used to prevent transnitrosation of R-SNOs. Development of plasma Alb-SNO below 300 nM was observed after intravenous injection (i.v.) of nitric oxide (NO) solution (0.1 to 3 ml/g), NOC7 (an NO releasing agent, above 1 µg/kg), and a low dose of Alb-SNO (10 nmol/kg). However, blood pressure was not significantly reduced by NO solution or Alb-SNO. Intravenous injection of a high dose of Alb-SNO (300 nmol/kg) significantly reduced blood pressure with the appearance not only Alb-SNO in micromolar level in plasma, but also G-SNO in lesser degree. Conversely, the hypotensive effect of Cys-NO (300 nmol/kg, i.v.) and G-SNO (300 nmol/kg, i.v.) accompanied development of Alb-SNO (micromolar level), but not Cys-NO or G-SNO in plasma. R-SNOs were not found in plasma during profound hypotension induced by acetylcholine (10 and 30 µg/kg/min, continuous i.v.), glyceryl trinitrate (100 µg/kg, i.v.), sodium nitroprusside (100 µg/kg, i.v.), and isosorbide dinitrate (300 µg/kg, i.v.). These results indicate that R-SNOs do not play an important role under unstimulated condition. In addition, plasma R-SNOs may not be involved in pharmacological vasodilation where contributions of NO or R-SNOs are suggested.

Different disappearance rates of plasma nitrite (NO(2)(-)) contribute to apparent steady-state arterio-venous differences in anesthetized animals.

A possible cause of arterio-venous (A-V) differences in plasma nitrite (NO(2)(-)) levels under steady-state conditions and kinetic features of NO(2)(-) in arterial and venous blood were examined. In isolated rabbit blood, plasma NO(2)(-) in venous blood disappeared faster than that in arterial blood and was accompanied by a concomitant increase in nitrate (NO(3)(-)), implicating oxidation as the main pertinent metabolic pathway. When data were corrected with respective elimination constants and time durations before plasma separation, no A-V difference was estimated under steady-state. Even after these corrections for NO(2)(-) loading in anesthetized rabbits, a large A-V difference in NO(2)(-) levels (arterial venous) was observed, followed by an exponential decrease in NO(2)(-) levels without a reciprocal increase in NO(3)(-) levels. There was a marked difference in NO(2)(-) decay between in vivo and ex vivo experiments, but no increases in the circulating blood were detected for other substances derived from NO(2)(-), such as methemoglobin or low- and high-molecular weight nitrosothiols. In rats and guinea pigs, absence and presence of the A-V difference were detected under steady-state conditions and after NO(2)(-) loading, respectively. These observations indicate that apparent A-V differences under steady-state are artifacts arising from different rates of NO(2)(-) disappearance in arterial versus venous plasma during sample handling, and that tissue compartments may contribute to changes in NO(2)(-) levels in circulating blood. Therefore, caution is required when evaluating plasma NO(2)(-) levels, especially in venous blood.

A study of cardiovascular function in Tsumura Suzuki obese diabetes, a new model mouse of type 2 diabetes.

Diabetes mellitus is a well known and important risk factor for cardiovascular diseases, including heart failure. A new model of Type 2 diabetes, Tsumura Suzuki Obese Diabetes (TSOD) mice, was introduced recently into the research field of diabetes. The cardiac functions of TSOD mice were studied in comparison with Tsumura Suzuki Non Obesity (TSNO, non-diabetic control) mice, for the first time. In vivo cardiovascular functions were measured by echocardiography and cardiac catheterization at 7, 12 and 18 months old. TSOD mice had no deterioration of cardiac function despite the long-term persistence of severe obesity, hyperglycemia, hyperinsulinemia and hyperlipidemia, including high density lipoprotein (HDL)-cholesterol. No histopathological abnormalities were observed in the heart of TSOD mice, while several histological abnormalities were observed in the pancreas and kidney of TSOD mice. To investigate vascular endothelium function at 7 months old, intravenous injection of acetylcholine (ACh; 1, 3, 10 microg/kg)- and N(G)-nitro-L-arginine methyl ester (L-NAME; 50 mg/kg)-induced mean blood pressure (BP) changes were used. ACh decreased whereas L-NAME increased BP, and no significant differences in BP changes were observed between TSOD and TSNO mice. Moreover, ACh-induced relaxation of the thoracic aortae isolated from TSOD and TSNO mice with intact endothelium were not significantly different. These findings suggest that vascular endothelial cells in TSOD mice are not impaired. It was clearly demonstrated that despite obvious diabetes, cardiac functions of TSOD mice were not impaired even at 18 months old.

Functional proteomic analysis of experimental autoimmune myocarditis-induced chronic heart failure in the rat.

Experimental autoimmune myocarditis (EAM)-induced heart failure in rats is used to study the pathogenesis of heart failure. Based on a proteomic analysis of soluble (S) and membranous (M) fractions extracted from ventricles of rats with a stable chronic form of EAM-induced heart failure, we assessed changes in protein levels and their correlation to heart functions to gain insights into the pathogenesis and to explore new targets for the treatment of heart failure. Proteins were separated by two-dimensional gel electrophoresis and silver stained spots were analyzed. In the S-fraction, 274+/-3 spots were detected in the normal (N)-group and 273+/-6 in the heart failure (HF)-group. In the HF-group, 26 of the spots were increased and 15 were decreased in intensity. In the M-fraction, 277+/-3 spots were detected in the N-group and 277+/-2 in the HF-group, with 20 spots increased and 10 decreased in intensity. We analyzed relationships between the expression of these proteins and 11 parameters of heart function, and found all the significantly changed spots to correlate with at least one of the parameters. We analyzed 49 spots that correlated with over 9 parameters of heart function using mass spectrometry, and identified 15 as proteins with increased expression including glucose regulated protein (GRP)78, an endoplasmic-stress related protein, and heat shock protein (HSP)90beta, a molecular chaperone, and 4 spots as proteins with decreased expression. It is suggested that in the heart failure model, GRP78 and HSP90beta play a role in the protection or deterioration of the heart and may be new targets for treatment.

Shikonin directly inhibits nitric oxide synthases: possible targets that affect thoracic aorta relaxation response and nitric oxide release from RAW 264.7 macrophages.

Recently, an isomeric mixture of herbal anti-inflammatory naphthoquinones shikonin and alkannin, and their derivatives, have been found to impair cellular responses involving nitric oxide (NO) and NO synthesis, like the acetylcholine-induced relaxation response of rat thoracic aorta and NO release from murine RAW 264.7 macrophages. However, the mechanisms of such effects, including whether NO synthase (NOS) activity is affected, remained unclear. We herein investigate possible targets of shikonin in these NOS-related events. Shikonin by itself dose-dependently inhibited the rat thoracic aorta relaxation in response to acetylcholine (pD'(2) value: 6.29). Its optical enantiomer, alkannin, was equally inhibitory in the aorta relaxation-response assay. In RAW 264.7 cells, shikonin inhibited the lipopolysaccharide-induced NO production by 82% at 1 microM. A cell-free assay to verify direct effects on NOS activity showed that shikonin inhibits all isoforms of NOS (IC(50)s, 4 - 7 microM), suggesting NOS as an inhibition target in both the events. Further possible targets of shikonin that might be involved in the inhibitions of the acetylcholine-induced aorta relaxation response and the NO generation by RAW 264.7 cells are also discussed. It is shown for the first time that shikonin inhibits NOS activity.

Arteriovenous differences in NO2- kinetics in anesthetized rabbits.

Despite of the importance of plasma NO2- as an index of endothelial nitric oxide (NO) formation and a substrate for NO production, only a limited kinetic knowledge is available in vivo. To address this issue, we intravenously injected NaNO2 into anesthetized rabbits and quantified changes in arterial and venous plasma NO2- levels. Plasma NO2- levels in arterial blood (956 +/- 220 nM) were slightly, but significantly, higher than those in venous blood (889 +/- 214 nM) under control conditions. Although similar half-lives of the NO2- were observed in arterial and venous plasma, significant arteriovenous differences were observed in the volume of distribution of the central compartment (0.158 +/- 0.007 l/kg in arterial blood and 0.295 +/- 0.015 l/kg in venous blood) and the peripheral compartment (0.259 +/- 0.035 l/kg in arterial blood and 0.135 +/- 0.034 l/kg in venous blood). When NOR1 (authentic NO) was injected, increases in NO2- levels were greater in arterial plasma, and decreases in blood pressure significantly correlated with arterial NO2- levels only (r = 0.90, p < 0.01). These results indicate that changes in NO2- are larger and more easily detectable as an index of NO formation in arterial plasma.

Quantifying nanomolar levels of nitrite in biological samples by HPLC-Griess method: special reference to arterio-venous difference in vivo.

Nitrite (NO(2)(-)) is assumed to play an important role in regulation of vascular tone as a reservoir of nitric oxide (NO). To examine its physiological contribution, however, a sensitive method is required for determination of the true level of NO(2)(-) in biological samples. To this end, practical consideration to avoid NO(2)(-) contamination through the quantification procedure is important. We present here a highly sensitive and accurate method for determining NO(2)(-) in plasma by improving the HPLC-Griess system with minimal NO(2)(-) contamination in the samples. The system achieved high sensitivity (detection limit of 2 nM and sensitivity to 1 nM) and complete separation of the NO(2)(-) signal peak by modifying the system setup and mobile phase. Using this method, we achieved acceptable quantification of low NO(2)(-) levels in plasma. Deproteinization by ultrafiltration and exposure to atmosphere before measurement were identified as the major sources of NO(2)(-) contamination during sample processing. We addressed these issues by the use of methanol for deproteinization and gas-tight caps. These countermeasures allowed us to detect small arterio-venous NO(2)(-) differences in rabbit plasma that may indicate kinetic difference of NO(2)(-) in a small number of samples (n = 6). This difference became prominent when NO(2)(-) or a NO releasing agent, NOR1, was intravenously applied. Our results indicate that application of a sensitive method with careful handling is important for accurate determination of NO(2)(-) and that our method is applicable for further examination of the kinetic features of NO(2)(-) in vivo.

Which skeletal myoblasts and how to be transplanted for cardiac repair?

Clinical efficacy of skeletal myoblast (skMb) transplantation is controversial whether this treatment produces beneficial outcome in patients with dilated cardiomyopathy (DCM). Based on immunological tolerance between wild-type and DCM hamsters with the deletion of delta-sarcoglycan (SG) gene, skMb engraftment in TO-2 myocardium (3x10(5) cells in approximately 100mg heart) was verified by the donor-specific expression of delta-SG transgene constitutively produced throughout myogenesis. At 5 weeks after the transplantation, the cell rates expressing fast-myosin heavy chain (MHC) exceeded slow-MHC in delta-SG(+) cells. Fifteen weeks after (corresponding to approximately 12 years in humans), fast MHC(+) cells nullified, but the delta-SG(+) and slow MHC(+) cell number remained unaltered. These skMbs fused with host cardiomyocytes via connexin-43 and intercalated disc, modestly improving the hemodynamics without arrhythmia, when engrafted skMbs were sparsely disseminated in autopsied myocardium. These results provide us evidence that disseminating delivery of slow-MHC(+) myoblasts is promising for repairing DCM heart using histocompatible skeletal myoblasts in future.

A novel scheme of dystrophin disruption for the progression of advanced heart failure.

The precise mechanism of the progression of advanced heart failure is unknown. We assessed a new scheme in two heart failure models: (I) congenital dilated cardiomyopathy (DCM) in TO-2 strain hamsters lacking delta-sarcoglycan (SG) gene and (II) administration of a high-dose of isoproterenol, as an acute heart failure in normal rats. In TO-2 hamsters, we followed the time course of the histological, physiological and metabolic the progressions of heart failure to the end stage. Dystrophin localization detected by immunostaining age-dependently to the myoplasm and the in situ sarcolemma fragility evaluated by Evans blue entry was increased in the same cardiomyocytes. Western blotting revealed a limited cleavage of the dystrophin protein at the rod domain, strongly suggesting a contribution of endogenous protease(s). We found a remarkable up-regulation of the amount of calpain-1 and -2, and no change of their counterpart, calpastatin. After supplementing TO-2 hearts with the normal delta-SG gene in vivo, these pathological alterations and the animals' survival improved. Furthermore, dystrophin but not delta-SG was disrupted by a high dose of isoproterenol, translocated from the sarcolemma to the myoplasm and fragmented. These results of heart failure, irrespective of the hereditary or acquired origin, indicate a vicious cycle formed by the increased sarcolemma permeability, preferential activation of calpain over calpastatin, and translocation and cleavage of dystrophin would commonly lead to advanced heart failure.

A novel paradigm for therapeutic basis of advanced heart failure--assessment by gene therapy.

The precise mechanism(s) of the progression of advanced heart failure (HF) should be determined to establish strategies for its treatment or prevention. Based on pathological, molecular, and physiological findings in 3 animal models and human cases, we propose a novel scheme that a vicious cycle formed by increased sarcolemma (SL) permeability, preferential activation of calpain over calpastatin, and translocation and cleavage of dystrophin (Dys) commonly lead to advanced HF. The aim of this article was to assess our recent paradigm that disruption of myocardial Dys is a final common pathway to advanced HF, irrespective of its hereditary or acquired origin, but not intended to provide a comprehensive overview of the various factors that may be involved in the course of HF in different clinical settings. In addition, each component of Dys-associated proteins (DAP) was heterogeneously degraded in vivo and in vitro, i.e. Dys and alpha-sarcoglycan (SG) were markedly destroyed using isolated calpain 2, while delta-SG was not degraded at all. The up-regulation of calpain 2 was confirmed through previously published data that remain insufficient for precise evaluation, supporting our new scheme that the activation of calpain(s) is involved in the steady process of Dys cleavage. In addition, somatic gene therapy is discussed as a potential option to ameliorate the physiological/metabolic indices and to improve the prognosis.

Translocation and cleavage of myocardial dystrophin as a common pathway to advanced heart failure: a scheme for the progression of cardiac dysfunction.

Advanced heart failure (HF) is the leading cause of death in developed countries. The mechanism underlying the progression of cardiac dysfunction needs to be clarified to establish approaches to prevention or treatment. Here, using TO-2 hamsters with hereditary dilated cardiomyopathy, we show age-dependent cleavage and translocation of myocardial dystrophin (Dys) from the sarcolemma (SL) to the myoplasm, increased SL permeability in situ, and a close relationship between the loss of Dys and hemodynamic indices. In addition, we observed a surprising correlation between the amount of Dys and the survival rate. Dys disruption is not an epiphenomenon but directly precedes progression to advanced HF, because long-lasting transfer of the missing delta-SG gene to degrading cardiomyocytes in vivo with biologically nontoxic recombinant adenoassociated virus (rAAV) vector ameliorated all of the pathological features and changed the disease prognosis. Furthermore, acute HF after isoproterenol toxicity and chronic HF after coronary ligation in rats both time-dependently cause Dys disruption in the degrading myocardium. Dys cleavage was also detected in human hearts from patients with dilated cardiomyopathy of unidentified etiology, supporting a scheme consisting of SL instability, Dys cleavage, and translocation of Dys from the SL to the myoplasm, irrespective of an acute or chronic disease course and a hereditary or acquired origin. Hereditary HF may be curable with gene therapy, once the responsible gene is identified and precisely corrected.

[A new paradigm for the progression of advanced heart failure].

To clarify the precise mechanism for the progression of advanced heart failure (AdHF), we assessed the scheme in two HF models, using (I) TO-2 strain hamsters sharing common genetic and clinical features to human families with the delta-sarcoglycan (SG) gene mutation and (II) administration of a high-dose (HD) of isoproterenol (Isp) to normal rats. Delta-SG is a component in dystrophin (Dys)-related proteins that stabilize the sarcolemma (SL) during repeated heart beats. In TO-2, we followed time course of hemodynamics, immunostaining and Western blotting of Dys and in situ SL permeability by Evans blue uptake with or without the gene therapy. Dys was age-dependently translocated from the SL to myoplasm (MP) where the SL instability accompanied the fragmentation of Dys. By gene therapy to supplement the normal delta-SG gene in hearts in vivo, we found that Dys translocation was selectively improved in cardiomyocytes expressing the delta-SG transgene, where the SL fragility was ameliorated. Most importantly, the survival period of the animals was prolonged. Furthermore, Dys but not delta-SG was also time-dependently shifted with a HD of Isp from the SL to MP and fragmented, while delta-SG was preserved intact. We present a novel paradigm that disruption of Dys, but not delta-SG per se, leads to AdHF irrespective of hereditary or acquired origin.

Efficacy of fluticasone propionate compared with beclomethasone dipropionate in bronchial asthma: improvement in compliance and symptoms by fluticasone.

Recent studies have shown that fluticasone propionate (FP) was more effective than beclomethasone dipropionate (BDP) inhalation even at a dose reduced by twofold or more in the treatment of bronchial asthma. Here, we further compared the effectiveness of FP and BDP, including rates of drug compliance. Forty-two symptomatic patients were treated by BDP (1000 +/- 345; mean +/- SD; microgram/day) for 8 weeks, followed by FP at one-half the respective dose, and peak expiratory flow and forced expiratory volume in 1 second were investigated. In addition, the patients were asked about drug compliance and factors related to compliance (expressed using a visual analogue scale). Significant increases of peak expiratory flow (from 316 +/- 96 L/minute to 345 +/- 86 L/minute) and forced expiratory volume in 1 second (from 1.7 +/- 0.5 L to 1.9 +/- 0.4 L) were found. Furthermore, significantly higher scores were obtained for compliance and various factors related to compliance. These data indicate that FP is more effective than a twofold higher dosage of BDP and that better compliance with the use of FP, probably because of improved various factors associated with FP compliance, contributes to FP efficacy.

Sarcolemmal fragility secondary to the degradation of dystrophin in dilated cardiomyopathy, as estimated by electron microscopy.

A common gene deletion or mutation of delta-sarcoglycan (delta-SG) in dystrophin-related proteins (DRPs) is identified in both TO-2 strain hamsters and human families with dilated cardiomyopathy. We have succeeded in the long-lasting in vivo supplementation of a normal delta-SG gene by recombinant adeno-associated virus vector, restoration of the morphological and functional degeneration, and improvement in the prognosis of the TO-2 hamster. To evaluate the integrity of the sarcolemma (SL) and the subsequent change of organelles in cardiomyocytes of the TO-2 strain hamster, we examined electron microscopy (EM) images focusing on the sarcolemmal stability at the end stage of heart failure. Two types of sarcolemmal degradation were detected: the widened and locally thickened SL, and blurred and discontinuous SL. Bizarrely formed mitochondria of varying sizes were also observed. Immuno-EM revealed clear expression of dystrophin in the SL and intense expression at the costamere as well as at the T-tubules in the control F1B strain hearts, but a patchy deposition of dystrophin was observed along the SL without the transgene of delta-SG. In contrast to the previous reports that dystrophin's integrity was intact, the present results suggest that the gene deletion of delta-SG and the loss of delta-SG protein in the SL cardioselectively cause the morphological and functional deterioration of dystrophin and the resultant instability of the SL. The sarcolemmal fragility may be similar to Duchenne-type progressive muscular dystrophy in skeletal muscle. In addition to the mechanical role, another aspect of DRPs for the intracellular signal transmission is also discussed.

[Somatic gene therapy of dilated cardiomyopathy].

The hereditary form of dilated cardiomyopathy (DCM) accounts for about 20% of human DCM and is a major cause of heart failure. TO-2 strain hamsters show DCM, a gene deletion of delta-sarcoglycan (SG), loss of all four SGs, alpha-, beta-, gamma- and delta-SG proteins, and are useful for developing gene therapy of the hereditary DCM. The delta-SG is a component of dystrophin-associated glycoprotein complex that stabilizes sarcolemma. Four familial and sporadic DCM cases have been reported in human patients with the same delta-SG gene mutation. To establish the potential gene therapy of DCM, efficient and long-lasting transduction of the responsible gene is mandatory, especially for improving the functional defect. Recombinant adeno-associated virus (rAAV) vector with delta-SG gene was intramurally transfected to the TO-2 hearts at 5-weeks-old. The transfected myocardium revealed robust expression of both transcript and transgene after 10 and 20 weeks. Immunohistological analyses demonstrated re-expression of not only delta-SG but also the other three SGs and normalization of the diameter of transduced cardiomyocytes without the pathogenicity. Hemodynamic studies revealed preferential amelioration of the diastolic indices. It suggests a novel strategy for the treatment of DCM and the rAAV vector is available for the treatment of several human diseases because of its safety and efficacy.

Rescue of hereditary form of dilated cardiomyopathy by rAAV-mediated somatic gene therapy: amelioration of morphological findings, sarcolemmal permeability, cardiac performances, and the prognosis of TO-2 hamsters.

The hereditary form comprises approximately 1/5 of patients with dilated cardiomyopathy (DCM) and is a major cause of advanced heart failure. Medical and socioeconomic settings require novel treatments other than cardiac transplantation. TO-2 strain hamsters with congenital DCM show similar clinical and genetic backgrounds to human cases that have defects in the delta-sarcoglycan (delta-SG) gene. To examine the long-term in vivo supplement of normal delta-SG gene driven by cytomegalovirus promoter, we analyzed the pathophysiologic effects of the transgene expression in TO-2 hearts by using recombinant adeno-associated virus vector. The transgene preserved sarcolemmal permeability detected in situ by mutual exclusivity between cardiomyocytes taking up intravenously administered Evans blue dye and expressing the delta-SG transgene throughout life. The persistent amelioration of sarcolemmal integrity improved wall thickness and the calcification score postmortem. Furthermore, in vivo myocardial contractility and hemodynamics, measured by echocardiography and cardiac catheterization, respectively, were normalized, especially in the diastolic performance. Most importantly, the survival period of the TO-2 hamsters was prolonged after the delta-SG gene transduction, and the animals remained active, exceeding the life expectancy of animals without transduction of the responsible gene. These results provide the first evidence that somatic gene therapy is promising for human DCM treatment, if the rAAV vector can be justified for clinical use.

Gene therapy prevents disruption of dystrophin-related proteins in a model of hereditary dilated cardiomyopathy in hamsters.

BACKGROUND: The TO-2 hamster is an animal model of dilated cardiomyopathy (DCM). It has genetic and clinical features in common with humans who carry the gene deletion or mutation of the delta-sarcoglycan (SG) gene, a component in dystrophin-related proteins (DRP). DRP stabilise the sarcolemma during cardiac contraction. We performed in vivo gene therapy of the TO-2 hamster, whose heart is defective in all four SG proteins, to determine its potential as a model for therapy for DCM. In addition to the hereditary origin, heart failure is aggravated by treatment with catecholamines and ameliorated by the administration of some kinds of beta-antagonist both in humans and in TO-2 hamsters. METHODS: Gene therapy for DCM was achieved by supplementing the delta-SG gene with rAAV vector and intramurally delivering rAAV-delta-SG into the cardiac apex and left ventricle. RESULTS: This treatment resulted in: (i) a sustained and non-pathogenic expression of both the transcript and transgene of delta-SG and all other SG proteins; (ii) improvement to both morphological and physiological deterioration; and (iii) rescued prognosis compared with untreated TO-2 hamsters, and TO-2 hamsters transfected with reporter gene alone. Another acute heart-failure model was prepared by high-dose isoproterenol treatment in Wistar rats, which resulted in: (i) translocation of dystrophin, but not delta-SG, from the cardiac sarcolemma to the myoplasm; and (ii) fragmentation of dystrophin, probably due to the activation of endogenous protease(s) or proteasome(s) that contributed to muscular dystrophy-like degeneration occurring specifically in cardiomyocytes. CONCLUSIONS: Both the TO-2 hamster and the isoproterenol-treated Wistar rat models commonly experience disruption of dystrophin or DRP. Targeting the responsible gene with the use of a potent vector may provide a novel strategy for the treatment of advanced heart failure.