Long-term but not short-term p38 mitogen-activated protein kinase inhibition improves cardiac function and reduces cardiac remodeling post-myocardial infarction

p38 mitogen-activated protein kinase (p38 MAPK) inhibition exerts beneficial effects on left ventricular (LV) remodeling and dysfunction. p38 MAPK activity is transiently increased soon after myocardial infarction (MI), suggesting brief inhibition may afford the same benefit as long-term inhibition. We examined chronic 12-week p38 MAPK inhibition compared with short-term (7-day) inhibition, and then we discontinued inhibition after MI. Post-MI rats at day 7 received either vehicle, 4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-3-butyn-1-ol (RWJ67657; RWJ) for 12 weeks (long term; LT-RWJ), RWJ for 1 week and discontinued for 11 weeks (1-week RWJ), or continuous ramipril for 12 weeks. In separate groups of animals, 24 h after MI, vehicle or RWJ was administered for 7 days. Cardiac function was assessed by echocardiography and hemodynamic measurements. Percentage of fractional shortening improved after LT-RWJ and ramipril, but not after 1-week RWJ treatment. Likewise, LV contractility and maximal first derivative of left ventricular pressure (dP/dt(max)) was improved (12.5 and 14.4%) and LV end diastolic pressure (LVEDP) was reduced (49.4 and 54.6%) with both treatments. Functional outcomes were accompanied by regression of interstitial collagen I and alpha-smooth muscle actin expression in LV noninfarct, border, and infarct regions with LT-RWJ and ramipril treatment. Hypertrophy was reduced in noninfarct (18.3 and 12.2%) and border regions (16.3 and 12.0%) with both treatments, respectively. Animals receiving RWJ 24 h after MI for 7 days showed similar improvements in fractional shortening, dP/dt(max), LVEDP, including reduced fibrosis and hypertrophy. In vitro experiments confirmed a dose-dependent reduction in hypertrophy, with RWJ following tumor necrosis factor-alpha stimulation. Continuous but not short-term p38 MAPK blockade attenuates post-MI remodeling, which is associated with functional benefits on the myocardium.     

Adenylylcyclase gene transfer increases function of the failing heart

A persistent question in cardiovascular gene transfer concerns whether an exogenously delivered gene can increase function of the failing heart. Here we test the hypothesis that intracoronary delivery of adenovirus encoding adenylylcyclase type VI (Ad.ACVI) in the setting of active heart failure will increase function of the failing heart. As a model of heart failure, we used transgenic mice with dilated and poorly functioning hearts resulting from cardiac-directed expression of Galphaq.Galphaq mice with equivalent pretreatment impairment in left ventricular (LV) function (echocardiography) received 2.5x1010 viral particles of Ad.ACVI or Ad.EGFP (enhanced green fluorescent protein), or saline, by indirect intracoronary delivery. Serial echocardiograms obtained before and 14 days after gene transfer showed that Ad.ACVI increased LV ejection fraction (p<0.01) and velocity of circumferential fiber shortening (p<0.03). Detailed measurements in isolated hearts showed that ACVI gene transfer increased LV positive dP/dt (p=0.02) and LV negative dP/dt (p=0.01). Gene transfer was confirmed by polymerase chain reaction. These data show that, in an animal model that mimics key aspects of clinical congestive heart failure, cardiac gene transfer of ACVI increases function of the failing heart.     

Intravenous infusion of bone marrow mesenchymal stem cells improves myocardial function in a rat model of myocardial ischemia

OBJECTIVE: We investigated the effects of three different sites for delivery of bone marrow mesenchymal stem cells (MSCs) in a rat model of myocardial ischemia. DESIGN: Prospective, randomized, controlled study. SETTING: University affiliated research institute. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: A thoracotomy was performed under general anesthesia. Myocardial ischemia was induced by ligation of the left anterior descending coronary artery. One month later, animals were randomized to receive 5 x 10(6) MSCs labeled with PKH26 in phosphate buffer solution or phosphate buffer solution alone as a placebo by injection into right femoral vein, directly into the left ventricular (LV) cavity, or into the ischemic zone in the anterior ventricular free wall. MEASUREMENTS AND MAIN RESULTS: Echocardiographically measured myocardial function, including ejection fraction and fractional shortening, was quantitated 2 wks and 4 wks after administering MSCs or phosphate buffer solution. Hemodynamics, including cardiac index, LV dP/dt40, LV negative dP/dt, and LV diastolic pressure were measured 4 wks after administering MSCs or phosphate buffer solution. MSCs were counted in 5-microm sections obtained with cryostat from each harvested heart. Significant improvements in ejection fraction, fractional shortening, cardiac index, LV dP/dt40, LV negative dP/dt, and LV diastolic pressure followed injection of MSCs, regardless of the site of injection. However, the number of MSCs counted in the heart sections was significantly greater after direct myocardial injection. CONCLUSIONS: Independently of the site of injection and regardless of the different concentration of bone marrow mesenchymal stem cells identified in the myocardium, myocardial function was comparably improved in all groups of animals treated with MSCs.     

Echo doppler assessment of left ventricular function in rats with hypertensive hypertrophy.

OBJECTIVE: This study attempted to establish echocardiographic measurements of left ventricular (LV) mass and LV systolic and diastolic function, particularly in rats with hypertensive heart. METHODS: M-mode LV echograms and Doppler mitral flow were obtained in Dahl salt-sensitive rats placed on 0.3% or 8% sodium chloride diet. Echo Doppler measurements were compared with catheterization and pathologic measurements in 54 rats for LV mass and in 45 rats for LV systolic and diastolic function. RESULTS: Echocardiographic measurement of LV mass correlated well with pathologic measurement (r = 0.94, P <.01, n = 54, SEE = 0.08 mg), independent of LV size, aging, and therapeutic intervention. Endocardial fractional shortening (FS) correlated with LV peak + dP/dt (r = 0.56, n = 45, P <.01), and the correlation was improved to r = 0.71 if 11 rats with marked LV hypertrophy were excluded. Midwall FS correlated well with LV peak + dP/dt (r = 0.72, n = 45, P <.01) even if rats with extremely thickened ventricular wall were included. If midwall FS was lower than 14%, LV systolic dysfunction was very likely (sensitivity 67%, specificity 91%). Association of mitral E/A ratio of 2.0 or greater with deceleration time of shorter than 35 ms was an accurate indicator of elevated LV end-diastolic pressure (sensitivity 82%, specificity 86%) and increased lung weight because of congestive heart failure (sensitivity 89%, specificity 96%) in rats with hypertension. CONCLUSION: LV mass, LV systolic function, and LV end-diastolic pressure were assessable with echo Doppler in rats with hypertensive heart.     

Cardiac hypertrophy induced by thyroid hormone is independent of loading conditions and beta adrenoceptor blockade

This study was designed to determine whether thyroid hormone (T4) produces cardiac hypertrophy and alters ventricular function by direct effects on the heart or by alterations in adrenergic stimulation or changes in the peripheral circulation. Rats were treated with captopril (4 mg/ml of drinking water), propranolol (0.5 mg/ml of drinking water), hydralazine (80 mg/l of drinking water) or the combination of captopril and propranolol with and without T4 (15 micrograms/100 g b.w. i.p.). After 10 days, T4 increased (P less than .01) heart rate, left ventricular (LV) dP/dt and LV weight/body weight, but did not alter LV systolic pressure (SP) or enddiastolic pressure (EDP). Compared to treatment with T4 alone, captopril plus T4 decreased LV SP (P less than .05) and LV EDP (P less than .01); however, heart rate, LV dP/dt and LV weight/body weight were unchanged. Treatment with T4 plus propranolol decreased heart rate and LV EDP (P less than .05) compared to T4 alone; however, LV SP, LV dP/dt and LV weight/body weight were unchanged (P greater than .05). Hydralazine did not alter (P greater than .05) heart rate, LV SP, LV EDP or prevent the development of increased LV weight/body weight when given with T4; however, LV dP/dt was slightly decreased (P less than .05). Treatment with the combination of captopril and propranolol did not alter (P greater than .05) heart rate, LV SP, LV EDP or LV dP/dt and also failed to prevent the development of increased LV weight/body weight and LV dP/dt when given with T4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunosuppression decreases inflammation and increases AAV6-hSERCA2a-mediated SERCA2a expression

The calcium pump SERCA2a (sarcoplasmic reticulum calcium ATPase 2a), which plays a central role in cardiac contraction, shows decreased expression in heart failure (HF). Increasing SERCA2a expression in HF models improves cardiac function. We used direct cardiac delivery of adeno-associated virus encoding human SERCA2a (AAV6-hSERCA2a) in HF and normal canine models to study safety, efficacy, and the effects of immunosuppression. Tachycardic-paced dogs received left ventricle (LV) wall injection of AAV6-hSERCA2a or solvent. Pacing continued postinjection for 2 or 6 weeks, until euthanasia. Tissue/serum samples were analyzed for hSERCA2a expression (Western blot) and immune responses (histology and AAV6-neutralizing antibodies). Nonpaced dogs received AAV6-hSERCA2a and were analyzed at 12 weeks; a parallel cohort received AAV-hSERCA2a and immunosuppression. AAV-mediated cardiac expression of hSERCA2a peaked at 2 weeks and then declined (to ~50%; p<0.03, 6 vs. 2 weeks). LV end diastolic and end systolic diameters decreased in 6-week dogs treated with AAV6-hSERCA2a (p<0.05) whereas LV diameters increased in control dogs. Dogs receiving AAV6-hSERCA2a developed neutralizing antibodies (titer ≥1:120) and cardiac cellular infiltration. Immunosuppression dramatically reduced immune responses (reduced inflammation and neutralizing antibody titers <1:20), and maintained hSERCA2a expression. Thus cardiac injection of AAV6-hSERCA2a promotes local hSERCA2a expression and improves cardiac function. However, the hSERCA2a protein level is reduced by host immune responses. Immunosuppression alleviates immune responses and sustains transgene expression, and may be an important adjuvant for clinical gene therapy trials.     

Atorvastatin enhances interleukin-10 levels and improves cardiac function in rats after acute myocardial infarction

LV (left ventricular) remodelling is the basic mechanism of HF (heart failure) following MI (myocardial infarction). Although there is evidence that pro-inflammatory cytokines [including TNF-alpha (tumour necrosis factor-alpha) and IL-6 (interleukin-6)] are involved in the remodelling process, only little is known about the role of anti-inflammatory cytokines, such as IL-10. As accumulating evidence has revealed that statins possess anti-inflammatory properties, the aim of the present study was to elucidate the effect of atorvastatin on the modulation of the anti-inflammatory cytokine IL-10 and its effect on LV function in rats with HF subsequent to MI. Rats with MI, induced by permanent LAD (left anterior descending) branch coronary artery ligation, were treated for 4 weeks with atorvastatin (10 mg x kg(-1) of body weight x day(-1) via oral gavage) starting on the first day after induction of MI. Cardiac function was assessed by echocardiography and cardiac catheterization 4 weeks after MI induction. Membrane-bound and soluble fractions of TNF-alpha, IL-6 and IL-10 protein, the TNF-alpha/IL-10 ratio, serum levels of MCP-1 (monocyte chemoattractant protein-1) as well as myocardial macrophage infiltration were analysed. Treatment with atorvastatin significantly improved post-MI LV function (fractional shortening, +120%; dP/dt(max), +147%; and LV end-diastolic pressure, -27%). Furthermore atorvastatin treatment markedly decreased the levels of TNF-alpha, IL-6 and MCP-1, reduced myocardial infiltration of macrophages and significantly increased myocardial and serum levels of the anti-inflammatory cytokine IL-10. Thus the balance between pro-inflammatory and anti-inflammatory cytokines was shifted in the anti-inflammatory direction, as shown by a significantly decreased TNF-alpha/IL-10 ratio. Atorvastatin ameliorated early LV remodelling and improved LV function in rats with HF subsequent to MI. Our study suggests that the modulation of the balance between pro- and anti-inflammatory cytokines towards the anti-inflammatory cytokine IL-10 is one salutary mechanism underlying how atorvastatin influences post-MI remodelling and thus improves LV function.     

Depression of systolic and diastolic myocardial reserve during atrial pacing tachycardia in patients with dilated cardiomyopathy.

Previous reports have shown that increases in heart rate may result in enhanced left ventricular (LV) systolic and diastolic performance. To assess whether this phenomenon occurs in the presence of depressed LV function, the effects of pacing on LV pressure and volume were compared in seven patients with dilated cardiomyopathy (LV ejection fraction 0.19 +/- 0.11) and six patients with no or minimal coronary artery disease (LV ejection fraction 0.69 +/- 0.11). Patients with normal LV function demonstrated significant increases in LV peak-positive dP/dt, LV end-systolic pressure-volume ratio, LV peak filling rate, and a progressive leftward and downward shift of their pressure-volume diagrams, compatible with increased contractility and distensibility in response to pacing tachycardia. There was no change in LV peak-negative dP/dt or tau. Patients with dilated cardiomyopathy, in contrast, demonstrated no increase in either LV peak-positive dP/dt or the end-systolic pressure-volume ratio, and absence of a progressive leftward shift of their pressure-volume diagrams. Moreover, cardiomyopathy patients demonstrated no increase in LV peak-negative dP/dt or LV peak filling rate and a blunted downward shift of the diastolic limb of their pressure-volume diagrams. Tau, as determined from a derivative method, became abbreviated although never reaching control values. We conclude that patients with dilated cardiomyopathy may demonstrate little or no significant enhancement in systolic and diastolic function during atrial pacing tachycardia, suggesting a depression of both inotropic and lusitropic reserve.     

Effects of differential blockade of the renin-angiotensin system in postinfarcted rats

The present study compared short-term effects of the AT(1)-receptor antagonist, irbesartan with the angiotensin-converting enzyme (ACE) inhibitor, enalapril on systemic haemodynamics and cardiac remodelling in post-myocardia-infarcted (MI) rats. MI Sprague-Dawley rats were orally treated for 4 weeks with irbesartan (50 mg/kg/day) or enalapril (10 mg/kg/day). Then, cardiac and systemic haemodynamics were measured. Compared with the sham-operated group, left ventricular end-diastolic pressure (LVEDP), diastolic pressure (LVDP), heart weight to body weight ratio were all significantly increased in the MI group while the LV contractility (dP/dt) and pulsatile arterial pressure were significantly reduced. Both drugs reduced the elevated LVEDP and LVDP and prevented cardiac hypertrophy. Furthermore, irbesartan attenuated the right shift of the pressure-volume curves, prevented postinfarction-induced increase in urinary cyclic guanosine monophosphate and reduced urinary aldosterone excretion. Although both drugs were able to prevent further cardiac hypertrophy and improved cardiac filling pressure, only irbesartan limited LV dilatation. These data indicate that blockade of the renin-angiotensin system at the level of AT1 receptors may have a better cardioprotective benefit than reducing angiotensin II levels by ACE inhibition.     

Invasive optimization of cardiac resynchronization therapy: role of sequential biventricular and left ventricular pacing

BACKGROUND: Aim of this invasive study was to characterize and quantify changes in left ventricular (LV) systolic function due to sequential biventricular pacing (BV) as compared to right atrial triggered simultaneous BV (BV(0)), LV, and right ventricular (RV) pacing in patients with congestive heart failure (CHF). METHODS: In 22 CHF patients, all in sinus rhythm, temporary multisite pacing was performed prior to implantation of a permanent system. LV systolic function was evaluated invasively by the maximum rate of LV pressure increase (dP/dt(max)). Sequential BV pacing was performed with preactivation of either ventricle at 20-80 ms. RESULTS: In comparison to RV pacing, LV and BV(0) pacing increased dP/dt(max) by 33.9 +/- 19.3% and 34.0 +/- 22.6%, respectively (P < 0.001). In 9 patients, optimized sequential BV pacing further improved dP/dt(max) by 8.5 +/- 4.8% compared to BV(0) (range 3.3-17.1, P < 0.05). In 10 patients exhibiting a PR interval < or =200 ms, LV pacing was either superior (n = 6) or equal to BV(0) pacing (n = 4). In these 10 patients, LV pacing yielded a 7.4 +/- 8.0% higher dP/dt(max) than BV(0) pacing (P < 0.05). CONCLUSIONS: Using sequential BV pacing, generally with LV preactivation, moderate improvements in LV systolic function can be achieved in selected patients. Baseline PR interval may aid in the selection of the optimum cardiac resynchronization therapy (CRT) mode, favoring LV pacing in patients with a PR interval < or =200 ms.     

Transthoracic Sensor for Noninvasive Assessment of Left Ventricular Contractility: Validation in A Minipig Model of Chronic Heart Failure

Background: Invasively measured left ventricular (LV) dP/dt is the accepted standard for measuring acute and chronic directional changes in LV contractility. Recently, we developed a noninvasive force sensor based on an accelerometer positioned on the chest, which measures the vibrations generated by isovolumic myocardial contraction. The aim of this paper was to compare noninvasive (accelerometer) versus invasive (LV dP/dt) indices of myocardial contractility in a chronic minipig model of pacing‐induced heart failure (HF). Comparative assessment was performed both at rest and following dobutamine infusion. Methods: In adult male minipigs (n = 6), LV contractility was simultaneously assessed both invasively (LV dP/dt, Millar catheter) and noninvasively (accelerometer) at rest and following dobutamine (up to 7.5 mcg/kg/min), both before and after development of HF by pacing the LV at 180 beats/min for 3 weeks. Results: Invasive and noninvasive assessments were obtained in 24 conditions (12 at rest and 12 after dobutamine infusion). Sensor‐based cardiac force changes were significantly related to positive peak LV dP/dt_max changes following dobutamine infusion both in normal (r = 0.88, P < 0.001) and failing heart (r = 0.89, P < 0.001). The force‐frequency relation showed a tight correlation between invasive and noninvasive assessment (r = 0.68, P = 0.02). Conclusions: The force‐frequency relation can be assessed noninvasively by a transthoracic sensor based on an accelerometer. The method can efficiently detect the development of resting dysfunction and the contractile reserve at different HF steps, with potential for wearable HF monitoring. (PACE 2010; 795–803)     

Contribution of Atrioventricular Synchrony to Left Ventricular Systolic Function in a Closed-Chest Canine Model of Complete Heart Block: Implications for Single-Chamber Rate-Variable Cardiac Pacing

This study assessed the impact of atrioventricular (AV) synchrony on characteristics of left ventricular (LV) systolic function during ventricular pacing over a wide heart rate range in a conscious closed-chest canine model of complete AV block. Ten healthy adult dogs underwent thoracotomy during which complete AV block was created by formaldehyde injection, and paired ultrasonic sonomicrometers were positioned on the LV anterior-posterior minor axis. Following recovery from surgery, peak and end-diastolic LV transmural pressure, maximum dP/dt, stroke work, end-diastolic minor axis dimension, and maximum velocity of shortening, were quantitated at heart rates of 80, 100, 120, 140, and 160 beats per minute (bpm) during both ventricular pacing alone and AV sequential pacing with increasing AV intervals (0, 50, 100, 150, 200, 250, and 300 ms). Over the heart rate range tested, parameters of LV systolic function did not differ significantly during ventricular pacing with or without AV synchrony. For example, during ventricular pacing alone maximum LV dP/dt varied from 2110 +/- 70 mmHg/s to 2463 +/- 567 mmHg/s, a range essentially identical to that observed in the presence of AV synchrony. On the other hand, although the impact on LV performance of varying AV interval from 0 to 300 ms was small, differences tended to become more pronounced at higher pacing rates. At 80 bpm, neither stroke work nor maximum LV dP/dt were affected by change in AV interval, while at heart rates greater than or equal to 120 bpm both stroke work and LV dP/dt tended to maximize at AV intervals of 50 and 100 ms and thereafter declined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Post‐ischaemic angiogenic therapy using in vivo prevascularized ascorbic acid‐enriched myocardial artificial grafts improves heart function in a rat model

Angiogenesis plays a key role in post‐ischaemic myocardial repair. We hypothesized that epicardial implantation of an ascorbic acid (AA)‐enriched myocardial artificial graft (MAG), which has been prevascularized in the recipients' own body, promotes restoration of the ischaemic heart. Gelatin patches were seeded with GFP–luciferase‐expressing rat cardiomyoblasts and enriched with 5 μm AA. Grafts were prevascularized in vivo for 3 days, using a renal pouch model in rats. The MAG patch was then implanted into the same rat's ischaemic heart following myocardial infarction (MI). MAG‐treated animals (MAG group, n = 6) were compared to untreated infarcted animals as injury controls (MI group, n = 6) and sham‐operated rats as healthy controls (healthy group, n = 7). In vivo bioluminescence imaging indicated a decrease in donor cell survival by 83% during the first week post‐implantation. Echocardiographic and haemodynamic assessment 4 weeks after MI revealed that MAG treatment attenuated left ventricular (LV) remodelling (LV end‐systolic volume, 0.31 ± 0.13 vs 0.81 ± 0.01 ml, p < 0.05; LV end‐diastolic volume 0.79 ± 0.33 vs 1.83 ± 0.26 ml, p < 0.076) and preserved LV wall thickness (0.21 ± 0.03 vs 0.09 ± 0.005 cm, p < 0.05) compared to the MI group. Cardiac output was higher in MAG than MI (51.59 ± 6.5 vs 25.06 ± 4.24 ml/min, p < 0.01) and comparable to healthy rats (47.08 ± 1.9 ml/min). Histology showed decreased fibrosis, and a seven‐fold increase in blood vessel density in the scar area of MAG compared to MI group (15.3 ± 1.1 vs 2.1 ± 0.3 blood vessels/hpf, p < 0.0001). Implantation of AA‐enriched prevascularized grafts enhanced vascularity in ischaemic rat hearts, attenuated LV remodelling and preserved LV function. Copyright © 2011 John Wiley & Sons, Ltd.     

Prediction of postoperative left ventricular systolic function with Doppler-derived dP/dt in patients with chronic aortic regurgitation.

To evaluate the value of Doppler-derived dP/dt as a predictor of postoperative left ventricular (LV) systolic function in patients with chronic aortic regurgitation, we evaluated 29 patients who underwent aortic valve replacement (n = 17) or valve repair. Doppler-derived dP/dt was determined from the continuous wave Doppler signal of the aortic regurgitation jet preoperatively. Preoperative LV ejection fraction (LVEF) and Doppler-derived dP/dt were 48 +/- 11% and 701 +/- 204 mm Hg/s, respectively. LVEF decreased to 43 +/- 12% at immediate postoperative period and improved to 54 +/- 11% at late postoperative period. In multivariate analysis, only dP/dt was an independent predictor of late postoperative LVEF (r = 0.59, P =.006). A dP/dt 相似文献   

Noninvasive assessment of left ventricular isovolumic contraction and relaxation with continuous wave Doppler aortic regurgitant velocity signals: an in vivo validation study.

The purpose of this study was to provide fundamental in vivo validation of a method with the use of aortic regurgitant (AR) jet signals recorded with continuous wave (CW) Doppler for assessing left ventricular (LV) isovolumic contraction and relaxation. Preliminary studies have suggested that analysis of CW Doppler AR velocity signals permits the estimation of LV positive and negative dP/dt. We studied 19 hemodynamically different states in 6 sheep with surgically induced chronic aortic regurgitation. CW AR velocity spectra and high-fidelity LV and aortic pressures were recorded simultaneously. Rates of LV pressure rise and fall (RPR and RPF) were calculated by determining the time interval between points at 1 m/s and 2.5 m/s in the deceleration and acceleration slopes of the CW Doppler AR velocity envelope (corresponding to a pressure change of 21 mm Hg). RPR and RPF calculated by CW Doppler analysis for each state were compared with the peak positive dP/dt and negative dP/dt, obtained from the corresponding high-fidelity LV pressure curve, respectively. The LV peak positive and negative dP/dt derived by catheter ranged from 817 to 2625 mm Hg/s and from 917 to 2583 mm Hg/s, respectively. Multiple regression analysis showed that Doppler RPR correlated well with catheter peak positive dP/dt (r = 0.93; mean differences, -413 +/- 250 mm Hg/s). There was also good correlation and agreement between Doppler RPF and the catheter peak negative dP/dt (r = 0.89; mean difference, -279 +/- 239 mm Hg/s). Both Doppler-determined RPR and RPF underestimated their respective LV peak dP/dt. CW Doppler AR spectra can provide a reliable noninvasive estimate of LV dP/dt and could be helpful in the serial assessment of ventricular function in patients with aortic regurgitation.     

Transgene expression after rep-mediated site-specific integration into chromosome 19

We have used a plasmid-based transfection model of the adeno-associated virus (AAV) Rep-mediated site-specific integration (RMSSI) pathway to characterize the stability and expression of a site-specifically integrated transgene (either green fluorescent protein [GFP] or chloramphenicol acetyltransferase [CAT]). Three plasmids containing the AAV p5 integration efficiency element (p5IEE) have been used to study integration and transgene expression in HeLa cells: (1) pRepGFP(itr+) contains both AAV ITRs, rep, and p5IEE and can be used as either a plasmid or rAAV vehicle for integration; (2) pRepGFP(itr-) contains the AAV rep gene and the p5IEE; (3) pAd-p5CAT contains only the 138-bp p5IEE of AAV. The data presented demonstrate that in the absence of drug selection, all three constructs undergo site-specific integration (efficiencies of between 10 and 40% of transduced cell lines). At 6 weeks posttransfection most cell lines that underwent RMSSI also expressed the appropriate transgene product. By 18 weeks posttransfection cell lines that were established with rep in cis to the transgene showed a decline in transgene expression as well as a loss of transgene DNA. In many cell lines, there appears to be transgene-containing DNA that does not contribute to gene expression. Data support a model of gene expression and transgene instability through a Rep-mediated pathway. In contrast to rep-containing cell lines, clonal cell lines containing p5IEECAT (with Rep provided in trans) maintained both the integrated transgene and transgene expression throughout the entire experimental time course (18 weeks).     

Validation of the myocardial performance index by echocardiography in mice: a noninvasive measure of left ventricular function.

BACKGROUND: The myocardial performance index (MPI) is a Doppler-based measure of left ventricular (LV) function. It is noninvasive, independent of LV shape, and does not require dimensional measurements. However, it has never been validated in mice. METHODS: A total of 29 anesthetized mice with LV pressure catheters underwent echocardiography (2-dimensional, M-mode, and Doppler) at baseline and during manipulations of beta-adrenergic tone, temperature, preload, and afterload. The maximum derivative of LV pressure with respect to time (dP/dt(max)) was compared with MPI, fractional shortening (FS), mean velocity of circumferential fiber shortening, and the FS/MPI ratio. RESULTS: MPI (baseline 0.44 +/- 0.07) correlated strongly with dP/dt(max) (R = -.779, P <.001), as did FS and mean velocity of circumferential fiber shortening. MPI differed significantly with contractility, preload, and afterload manipulation. FS/MPI showed the best correlation with dP/dt(max). CONCLUSIONS: MPI strongly correlates with dP/dt(max) over a range of hemodynamic conditions in mice. It can be used as a noninvasive index of LV function in this species.     

Effect of viral dose on neutralizing antibody response and transgene expression after AAV1 vector re-administration in mice

Neutralizing antibodies (nAB) at the time of administration hamper the effectiveness of adeno-associated virus (AAV) as a clinical DNA delivery system. The present study was designed to investigate if AAV re-administration in muscle tissue is dependent on the nAB titer. Recombinant (r)AAV serotype 1, as a promising candidate for targeting skeletal muscle, was used for gene delivery. C57Bl/6 mice were infected intramuscularly with doses between 1 x 10(9) and 5 x 10(10) virus particles (vp) of AAV1-expressing luciferase (AAV1-luc) or human interferon-beta (AAV1-hIFNbeta). Increasing transgene expression was observed over the first 2 months and anti-AAV1 nAB titers peaked between weeks 4 and 8. Six months after the first administration, 5 x 10(10) vp of AAV1-IFNbeta were re-administered. Following re-administration, nAB titers increased but did not significantly affect transgene expression from the AAV vector that had been administered first. In contrast, hIFNbeta expression originating from the second vector administration was significantly diminished and reflected the nAB titer present at the day of re-administration. The present study extends earlier observations that preexisting nAB affects AAV1 re-administration. The level of nAB is proportional to the virus dose used for the first injection and transgene expression following re-administration is dependent on preexisting nAB titer.     

Utility of PEGylated recombinant adeno-associated viruses for gene transfer.

Adeno-associated virus (AAV), capable of producing significant, long-term transgene expression, is one of the least toxic vectors employed in pre-clinical and clinical studies of gene transfer. One limitation is generation of neutralizing antibodies against viral capsids, blocking gene expression after readministration. AAV2 capsids were modified with poly(ethylene) glycols (PEGs) activated by cyanuric chloride (CCPEG), succinimidyl succinate (SSPEG) and tresyl chloride (TMPEG). SSPEG and TMPEG conjugation did not compromise gene transfer to the liver and muscle and improved gene expression in the lung 5 fold. Transduction efficiency of CCPEG-AAV was impeded in all tissues by aggregation. TMPEG afforded the best protection from neutralization in vitro and in vivo. Gene expression in mice immunized against unmodified AAV was reduced by a factor of 10 from that of na?ve animals after intramuscular rechallenge with PEGylated AAV but was not significantly different from na?ve mice after intravenous readministration (p=0.08). Gene expression was markedly reduced in muscle after two doses of PEGylated AAV. In contrast, mice given two intravenous doses of TMPEG-AAV had significantly higher transgene levels than na?ve animals 14 days after rechallenge (p=0.001). This technology could promote successful readministration of vector in the clinic and marked expression in patients with anti-AAV antibodies.     

Treatment of verapamil toxicity in intact dogs.

The treatment of verapamil toxicity was examined in lightly sedated dogs. Verapamil, administered as a bolus (0.72 mg/kg) followed by a continuous infusion (0.11 mg/kg per min), decreased cardiac output (CO) from 3.1 +/- 0.1 to 1.7 +/- 0.1 liter/min (P less than 0.001), heart rate (HR) from 85 +/- 4 to 57 +/- 3 beats/min (P less than 0.001), left ventricular derivative of pressure with respect to time (LV dP/dt) from 2,085 +/- 828 to 783 +/- 78 mm Hg/s (P less than 0.001), mean aortic pressure (AO) from 77 +/- 4 to 38 +/- 2 mm Hg (P less than 0.001) and stroke volume from 39 +/- 3 to 28 +/- 2 ml/beat (P less than 0.01). In verapamil-toxic animals isoproterenol increased HR, CO, LV dP/dt, and AO; calcium chloride increased LV dP/dt and AO; norepinephrine, epinephrine, and dopamine increased CO, AO, and LV dP/dt, atropine increased HR, CO, and AO. Phenylephrine (13-55 micrograms/kg per min) produced no changes except a small increase in AO while very high dose phenylephrine (300 micrograms/kg per min) increased AO, CO, and LV dP/dt. 4-Aminopyridine (4-AP) increased HR, CO, LV dP/dt, and AO. When administered prior to verapamil, 4-AP prevented the development of verapamil toxicity as shown by the significantly higher AO (P less than 0.001), CO (P less than 0.01), and LV dP/dt (P less than 0.01) when 4-AP followed by verapamil was compared to verapamil alone. In conclusion, there does not appear to be a single specific therapy for verapamil toxicity, however it can be partially corrected by presently available pharmacologic therapy and 4-AP.