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Background: Left ventricular assist device (LVAD) treatment is known to lead to structural and functional cellular modifications in the heart. The relevance of these changes for clinical recovery is unknown., Methods and Results: We compared properties of cardiomyocytes obtained from tissue taken at explantation of the LVAD in patients with clinical recovery with those obtained from hearts of patients who did not show clinical recovery, thus requiring transplantation. Compared with myocytes taken at implantation, both the recovery and nonrecovery groups showed approximately 50% reduction in cell capacitance, an index of cell size. However, action potential duration shortened, L-type Ca2+ current fast inactivation was more rapid, and sarcoplasmic reticulum Ca2+ content was increased in the recovery compared with the nonrecovery group., Conclusions: These results show that specific changes in excitation-contraction coupling, and not regression of cellular hypertrophy, are specifically associated with clinical recovery after LVAD and further identify sarcoplasmic reticulum Ca2+ handling as a key functional determinant in patients with heart failure.

Background: Upregulation of heat shock protein 70 (HSP70) is beneficial in cardioprotection against ischemia-reperfusion injury, but the mechanism of action is unclear. We studied the role of HSP70 overexpression through gene therapy on mitochondrial function and ventricular recovery in a protocol that mimics clinical donor heart preservation., Methods and Results: Hemagglutinating virus of Japan (HVJ)-liposome technique was used to transfect isolated rat hearts via intracoronary infusion of either the HSP70 gene (HSP group, n=16) or no gene (CON group, n=16), which was heterotopically transplanted into recipient rats. Four days after surgery, hearts were either perfused on a Langendorff apparatus for 30 minutes at 37 degrees C (preischemia studies [n=8/group]) or perfused for 30 minutes at 37 degrees C, cardioplegically arrested for 4 hours at 4 degrees C, and reperfused for 30 minutes at 37 degrees C (postischemia studies [n=8/group]). Western blotting and immunohistochemistry confirmed HSP70 upregulation in the HSP group. Postischemic mitochondrial respiratory control indices (RCIs) were significantly better preserved in HSP than in CON hearts: NAD(+)-linked RCI values were 9.54+/-1.1 versus 10.62+/-0.46 before ischemia (NS) but 7.98+/-0.69 versus 1.28+/-0.15 after ischemia (P<0.05), and FAD-linked RCI values were 6.87+/-0.88 versus 6.73+/-0.93 before ischemia (NS) but 4.26+/-0.41 versus 1.34+/-0.13 after ischemia (P<0.05). Postischemic recovery of mechanical function was greater in HSP than in CON hearts: left ventricular developed pressure recovery was 72.4+/-6.4% versus 59.7+/-5.3% (P<0.05), maximum dP/dt recovery was 77.9+/-6.6% versus 52.3+/-5.2% (P<0.05), and minimum dP/dt recovery was 72.4+/-7.2% versus 54.8+/-6.9% (P<0.05). Creatine kinase release in coronary effluent after reperfusion was 0.20+/-0.04 versus 0.34+/-0.06 IU. min(-1). g wet wt(-1) (P<0.05) in HSP versus in CON hearts., Conclusions: HSP70 upregulation protects mitochondrial function after ischemia-reperfusion injury; this was associated with improved preservation of ventricular function. Protection of mitochondrial function may be important in the development of future cardioprotective strategies.

Background: Interleukin-1 (IL-1) plays a role in mediating acute inflammation during ischemia-reperfusion (I/R) injury in the heart, which leads to both necrosis and apoptosis of cardiomyocytes. IL-1 receptor antagonist (IL-1ra) is known to inhibit the effects of IL-1alpha and IL-1beta, resulting in attenuated inflammatory injury, and to protect cells from IL-1beta-induced apoptosis in vitro. We hypothesized that IL-1ra overexpression would provide cardioprotection by reducing inflammation-mediated myocardial damage including apoptosis after I/R injury in vivo., Methods and Results: Rat hearts were transfected with human secreted-type IL-1ra gene by intracoronary infusion of Hemagglutinating Virus of Japan liposome and were heterotopically transplanted. IL-1ra overexpression in these hearts was confirmed by enzyme immunoassay and immunohistochemistry. Myocardial tolerance of the transplanted heart was evaluated with the use of a novel system in which the heart, existing within the recipient's abdomen, was given 30 minutes of ischemia by left coronary artery occlusion and 24 hours of reperfusion. Consequently, infarct size was decreased in IL-1ra-transfected hearts compared with control-transfected ones (26.9+/-3.2% versus 46.2+/-3.0%, P=0.001), corresponding to lower myocardial myeloperoxidase activity (2.20+/-0.69 versus 6.82+/-1.19 U/g wet wt, P<0.001) and decreased neutrophil infiltration in histological study. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and DNA-laddering studies demonstrated that cardiomyocyte apoptosis was attenuated in IL-1ra-transfected hearts (21.4+/-3.3 versus 41.4+/-3.4%, P=0.002), correlating with reduced post I/R upregulation of Bax, Bak, and caspase-3., Conclusions: IL-1ra introduced by gene transfection protected myocardium from I/R injury by attenuating the inflammatory response, which was associated with decreased apoptosis. This suggests a potentially important role of IL-1/IL-1ra in myocardial I/R injury and the value of IL-1ra-gene therapy for myocardial preservation.

Background: Vascular endothelial growth factor (VEGF) is a promising reagent for inducing myocardial angiogenesis. Skeletal myoblast transplantation has been shown to improve cardiac function in chronic heart failure models by regenerating muscle. We hypothesized that transplantation of VEGF-expressing myoblasts could effectively treat acute myocardial infarction by providing VEGF-induced cardioprotection through vasodilatation in the early phase, followed by angiogenesis effects in salvaging ischemic host myocardium combined with the functional benefits of newly formed, skeletal myoblast-derived muscle in the later phase., Methods and Results: Primary rat skeletal myoblasts were transfected with the human VEGF(165) gene using hemagglutinating virus of Japan-liposome with >95% transfection efficiency. Four million of these myoblasts (VEGF group), control-transfected myoblasts (control group), or medium only (medium group) was injected into syngeneic rat hearts 1 hour after left coronary artery occlusion. Myocardial VEGF-expression increased for 2 weeks in the VEGF group, resulting in enhanced angiogenesis without the formation of tumors. Grafted myoblasts had differentiated into multinucleated myotubes within host myocardium. Infarct size (33.3+/-1.4%, 38.1+/-1.4%, and 43.7+/-1.6% for VEGF, control, and medium groups, respectively; P=0.0005) was significantly reduced with VEGF treatment, and cardiac function improved in the VEGF group (maximum dP/dt: 4072.0+/-93.6, 3772.5+/-101.1, and 3482.5+/-90.6 mm Hg/s in the 3 groups, respectively; P=0.0011; minimum dP/dt: -504.2+/-68.5, -2311.3+/-57.0, and -2124.0+/-57.9 mm Hg/s, respectively; P=0.0008)., Conclusions: This combined strategy of cell transplantation with gene therapy could be of importance for the treatment of acute myocardial infarction.

Background: Adenosine (Ado) triggers numerous protective mechanisms in the heart that may attenuate ischemia-reperfusion injury in cardiac grafts. We aimed to establish whether sustained increase in endogenous Ado production by the combined application of Ado metabolism inhibitors and nucleotide precursors attenuates reperfusion injury in transplanted hearts., Methods and Results: Rat hearts were collected after the infusion of St Thomas' Hospital cardioplegic solution, stored at 4 degrees C for 4 hours, and heterotopically transplanted into the abdomen of recipient rats. A solution containing Ado deaminase inhibitor erythro-9(2-hydroxy-3-nonyl)adenine, Ado kinase inhibitor 5'-aminoadenosine, and nucleotide precursors adenine and ribose was administered at the time of reperfusion in the treated group, whereas saline was administered to control animals. After 1 or 24 hours, mechanical function of the transplanted hearts was evaluated in an ex vivo perfusion system followed by the determination of myocardial ATP with related metabolites and measurement of the activity of neutrophil-specific enzyme myeloperoxidase in cardiac homogenates. After 24 hours of reperfusion, maximum left ventricular developed pressure increased from 87.0+/-6.8 mm Hg (mean+/-SEM) in controls to 118.1+/-8.2 mm Hg in the treated group (P<0.05), ATP increased from 11.0+/-0.8 micromol/g dry wt in controls to 15.1+/-1.2 micromol/g dry wt in the treated group (P<0.01), and myeloperoxidase activity decreased from 2.23+/-0.60 U/g wet wt in controls to 0.58+/-0.12 U/g wet wt in the treated group (P<0.001). No differences in cardiac function, ATP, or myeloperoxidase activity were observed between the treated group and controls after 1 hour of reperfusion., Conclusions: The administration of Ado metabolism inhibitors with nucleotide precursors causes a sustained increase in endogenous Ado production and exerts a potent protective effect against reperfusion injury in transplanted hearts. Improved cardiac function and elevated ATP concentration were accompanied by complete amelioration of neutrophil infiltration in treated hearts, suggesting that reduction in postischemic inflammation could be an important mechanism of this protective effect.

Background: Molecular mechanisms underlying the deterioration of patients undergoing LV assist device (LVAD) implantation remain poorly understood. We studied the cytokines tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta and IL-6 and the terminal stage of the apoptotic pathway in patients with decompensating heart failure who required LVAD support and compared them with patients with less severe heart failure undergoing elective heart transplantation., Methods and Results: Myocardial and serum samples from 23 patients undergoing LVAD implantation were compared with those from 36 patients undergoing elective heart transplantation. Myocardial TNF-alpha mRNA (1.71-fold; P<0.05) and protein (3.43+/-0.19 versus 2.95+/-0.10 pg/mg protein; P<0.05) were elevated in the LVAD patients. Immunocytochemistry demonstrated TNF expression in the myocytes. Serum TNF-alpha was also elevated (12.5+/-1.9 versus 4.0+/-0.4 pg/mL; P<0.0001) in the LVAD patients. IL-6 mRNA (2.57-fold higher; P<0.005) and protein (27.83+/-9.35 versus 4.26+/-1.24 pg/mg protein; P<0.001) were higher in the LVAD candidates, as was serum IL-6 (79.3+/-23.6 versus 7.1+/-1.6 pg/mL; P<0.0001). Interleukin-1beta mRNA expression was 9.78-fold higher in the LVAD patients (P<0.001). iNOS mRNA expression was similar to that in advanced heart failure patients and was not further elevated in the LVAD patients. Levels of procaspase-9 (8.02+/-0.91 versus 6.16+/-0.43 oligodeoxynucleotide [OD] units; P<0.01), cleaved caspase-9 (10.02+/-1.0 versus 7.34+/-0.40 OD units; P<0.05), intact and spliced DFF-45 (4.58+/-0.75 versus 2.84+/-0.23 OD units; P<0.05) were raised in LVAD patients, but caspase-3 and human nuclease CPAN were not., Conclusions: Elevated TNF-alpha, IL-1beta, and IL-6 and alterations in the apoptotic pathway were found in the myocardium and elevated TNF-alpha and IL-6 in serum of deteriorating patients who required LVAD support. These occurrences may have therapeutic implications and influence the timing of LVAD insertion.

Background: The Ross operation has several theoretical advantages. However, concern exists regarding evolving pathology in the pulmonary homograft., Methods and Results: Consecutive patients (n=144; mean age 31 years, range 2 months to 64 years) undergoing the Ross operation were studied between 1993 and 2000. Echocardiographic examination of the pulmonary homograft was performed immediately after surgery, then at yearly intervals for a mean interval of 48 months. Fifteen patients (mean age 37 years) in whom echocardiography revealed peak pulmonary gradients >/=30 mm Hg (mean 46+/-18 mm Hg) underwent MRI with velocity mapping in a Picker 1.5-T magnet. No patient had more than mild pulmonary regurgitation. Four patients required reoperation for rapidly progressive pulmonary homograft stenosis; in all 4, there was macroscopic and microscopic evidence of a pronounced chronic adventitial reaction, with perivascular infiltration producing extrinsic compression. Freedom from any pulmonary homograft stenosis at 7-year follow-up was 79.7%, with instantaneous hazard falling to zero after 4 years. Freedom from reoperation at 7 years was 96.7%. In those studied with MRI, there was evidence of narrowing of the whole homograft or distal suture line in 14 of 15 patients, with obvious excess surrounding tissue in 11. Mean minimum diameter and peak velocity by MRI were 11+/-2 mm and 3.2+/-0.7 m/s, respectively. Multivariate analysis of patient-, surgery-, and homograft-related variables did not reveal any significant risk factors for development of neopulmonary stenosis., Conclusions: Pulmonary homograft stenosis after the Ross operation is clinically important and appears to represent an early postoperative inflammatory reaction to the pulmonary homograft that leads to extrinsic compression and/or shrinkage.

Background: Skeletal myoblast transplantation is promising for the treatment of end-stage heart failure. Direct intramyocardial injection is useful for local cell delivery but may not be effective in global dissemination of cells into the heart, which would be advantageous in treating generalized cardiac dysfunction as in dilated cardiomyopathy. We hypothesized that intracoronary infusion of myoblasts would disseminate cells more effectively, leading to functional improvement in global heart failure., Methods and Results: Heart failure was induced by the intraperitoneal administration of doxorubicin (total dose 15 mg/kg) in rat. One million primary skeletal myoblasts were then infused via the coronary arteries of an excised, failing doxorubicin-treated heart. After incubation under increased intracoronary pressure, the hearts were subsequently transplanted into syngeneic recipients. For the control group, doxorubicin-treated hearts were infused with medium only and transplanted. Four weeks after transplantation, Langendorff perfusion demonstrated that both maximum dP/dt (2797.6+/-103.3 versus 2326.9+/-133.1 mm Hg/s, P=0.01) and minimum dP/dt (-2067.4+/-88.1 versus -1718.8+/-91.3 mm Hg/s, P=0.02) were improved in myoblast-transplanted hearts compared with medium-infused hearts. This was associated with a sharper slope of the left ventricular developed pressure-volume curve and a reduced slope of the end-diastolic pressure-volume relation in the myoblast-transplanted hearts. Immunohistochemistry for skeletal myosin heavy chain showed that globally disseminated myoblasts had survived and differentiated into multinucleated myotubes that had aligned with the cardiac fiber axis within host myocardium. No significant myocardial infarction was observed., Conclusions: We demonstrated the feasibility and efficiency of skeletal myoblast transplantation via the intracoronary route as a promising strategy for improving cardiac function in global heart failure.

Background: The coxsackievirus and adenovirus receptor (CAR) was identified as a common cellular receptor for both viruses, but its biological and pathogenic relevance is uncertain. Knowledge of CAR localization in the human cardiovascular system is limited but important with respect to CAR-dependent viral infections and gene transfer using CAR-dependent viral vectors., Methods and Results: Explanted failing hearts from 13 patients (8 with dilated cardiomyopathy [DCM] and 5 with other heart diseases [non-DCM]) and normal donor hearts (n=7) were investigated for the expression levels and subcellular localization of CAR and the adenovirus coreceptors alpha(v)beta(3) and alpha(v)beta(5) integrins. CAR immunoreactivity was very low in normal and non-DCM hearts, whereas strong CAR signals occurred at the intercalated discs and sarcolemma in 5 of the 8 DCM hearts (62.5%); these strong signals colocalized with both integrins. In all hearts, CAR was detectable in subendothelial layers of the vessel wall, but not on the luminal endothelial surface, and on interstitial cells. Human CAR (hCAR) expressed in rat cardiomyocytes was targeted to cell-cell contacts, which resembled CAR localization in DCM hearts and resulted in 15-fold increased adenovirus uptake., Conclusions: Low hCAR abundance may render normal human myocardium resistant to CAR-dependent viruses, whereas re-expression of hCAR, such as that observed in DCM, may be a key determinant of cardiac susceptibility to viral infections. Asymmetric expression of hCAR in the vessel wall may be an important determinant of adenovirus tropism in humans. hCAR subcellular localization in human myocardium and hCAR targeting to cell-cell contacts in cardiomyocyte cultures suggest that hCAR may play a role in cell-cell contact formation.

Background: Prostacyclin has proved to be a beneficial treatment for patients with severe pulmonary hypertension. We postulated that the response may reflect, at least in part, inhibition of pulmonary artery smooth muscle cell (PASMC) growth., Methods and Results: Human PASMCs were derived from distal (<1-mm external diameter, n=8) and proximal (>8-mm external diameter, n=12) pulmonary arteries obtained at transplant surgery and pneumonectomy. The effects of the stable prostacyclin analogues on [methyl-(3)H]thymidine incorporation and cell proliferation were investigated by using immunohistochemically characterized cells. Distal cells proliferated faster than did proximal PASMCs and displayed a distinct sensitivity to cicaprost and iloprost. Both analogues inhibited thymidine uptake over 24 hours (20% to 60%, P<0.001; n=8) and abolished stimulation of DNA synthesis by platelet-derived growth factor-BB (10 ng/mL) in distal but not proximal cells. The inhibitory effect of cicaprost was mimicked by isoproterenol (10(-5) mol/L), forskolin (10(-5) mol/L), and dibutyryl cAMP (5x10(-4) mol/L) and was potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (5x10(-5) mol/L). Cicaprost (10(-10) to 10(-6) mol/L) inhibited the proliferation of PASMCs, which had been stimulated with either platelet-derived growth factor-BB or serum, and increased cAMP production. These effects were potentiated by 3-isobutyl-1-methylxanthine and attenuated by the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine (10(-5) to 10(-4) mol/L)., Conclusions: ++Cicaprost and iloprost inhibit DNA synthesis and proliferation to a greater extent in distal compared with proximal human PASMCs, acting at least in part via a cAMP-dependent mechanism. The results are consistent with the hypothesis that prostacyclin analogues inhibit vascular remodeling in pulmonary hypertension and demonstrate heterogeneity among human PASMCs.

Background: Myocardial dysfunction is a common and important problem in donor hearts. The mechanisms responsible remain unclear. We have studied the cytokines tumor necrosis factor (TNF)-alpha and interleukin-6 (IL-6) in the myocardium and serum from donors with myocardial dysfunction (unused donors) and compared them with donors with good ventricular function (used donors) and patients with advanced heart failure (HF)., Methods and Results: Clinical details and ventricular function were assessed in 46 donors (31 used, 15 unused). Real-time reverse transcription-polymerase chain reaction, Western blotting, and immunocytochemistry were performed on myocardium and immunoassays on serum. TNF-alpha mRNA was 1.6-fold higher in unused than in used donors (P:<0.005) and 1.74-fold higher than in 36 patients with HF. IL-6 mRNA was 2.4-fold higher in unused than in used donors (P:<0.0001) and 4.67-fold higher than in HF (P:<0.0001). Western blotting showed higher TNF-alpha in unused (218. 3+/-6.4, n=4 versus 187.3+/-5.4, n=3 OD units) than used donors (P:<0.05). TNF-alpha expression was localized to cardiac myocytes. Serum TNF-alpha was higher in unused (8.72+/-1.3 pg/mL, n=13) than in used (6.12+/-0.8 pg/mL, n=25, P:<0.05) donors and HF (4.0+/-0.4 pg/mL, n=17, P:<0.005). Serum TNF-alpha receptors did not differ between unused (4.3+/-0.8 and 8.6+/-1.6 ng/mL, n=10) and used (3. 5+/-0.4 and 6.5+/-1.1 ng/mL, n=24) donors. There was a trend for higher serum IL-6 in unused (16.5+/-2.9 pg/mL, n=9) compared with used (13.9+/-1.6 pg/mL, n=26, P:=NS) donors., Conclusions: This study documented an increase in the expression of TNF-alpha and IL-6 in the myocardium of all donor hearts that was more marked in the dysfunctional (unused) donor hearts. This was accompanied by similar changes in the serum. This might have important therapeutic implications.

Background: The purpose of this study was to determine whether T cells with indirect allospecificity could be detected in heart transplant recipients with chronic rejection., Method and Results: Human T-cell clones were used to determine the most effective way to deliver major histocompatibility complex alloantigens for indirect presentation. Seven allograft recipients with evidence of progressive, chronic rejection were selected. Four heart graft recipients with no evidence of chronic rejection were used as controls. Peripheral blood T cells and antigen-presenting cells from the recipients were cultured with frozen/thawed stored donor cells or major histocompatibility complex class I-derived synthetic peptides in limiting dilution cultures and then compared with controls using tetanus toxoid and frozen/thawed third-party cells with no human leukocyte antigens in common with the donor. In 5 of 7 patients analyzed who had chronic rejection, elevated frequencies of T cells with indirect, anti-donor specificity (iHTLf) were detected. No such elevated iHTLf were detected in recipients without chronic rejection., Discussion: iHTLf can be obtained from human transplant recipients, which supports the contention that the indirect pathway is involved in chronic transplant rejection.

Background: The surgical approach to aortic root aneurysm and/or dissection remains controversial. The use of valve-sparing operations, which are thought to have many advantages, is increasing. We hypothesized that the particular technique and type of surgery could influence valve motion characteristics and function. Therefore, we studied the instantaneous opening and closing characteristics of the aortic valve after the main 2 types of valve-sparing surgery., Methods and Results: In 20 patients (10 with tube replacement of the aortic root, group A; and 10 with separate replacement of the sinuses of Valsalva, group B) and 10 controls (group C), transthoracic and transesophageal studies on aortic valve dynamics were performed. Three distinct phases of aortic valve motion were identified. They were as follows: (1) a rapid opening, with a velocity of 20.9+/-4.2 cm/s in group C, 27.1+/-10.9 cm/s in group B (P=NS), and 58.3+/-18.4 cm/s in group A (group A versus group C, P<0. 001; group A versus group B, P=0.001); (2) a slow systolic closure, with 12.5+/-6.6% and 10.8+/-2.2% of maximal opening in groups C and B, respectively (P=NS), and 3.8+/-1.6% in group A (group A versus group C, P=0.001; group A versus group B, P<0.001); and (3) a rapid closing movement, with a velocity of 26.3+/-5.6 cm/s in group C, 32. 4+/-11.4 cm/s in group B (P=NS), and 21.8+/-3.5 cm/s in group A (group A versus group C, P=NS; group A versus group B, P=0.008). The pressure strain of the elastic modulus was different in groups C and B only at the commissures (682+/-145 g/cm(2) versus 1896+/-726 g/cm(2), respectively; P<0.001). At all root levels, the distensibility was reduced in group A (P<0.001). Systolic contact of aortic cusps and wall occurred only in group A., Conclusions: Near-normal opening and closing characteristics can be achieved by a technique that preserves the shape and independent mobility of the sinuses of Valsalva.