In human and experimental models of heart failure, sarcoplasmic reticulum Ca 2؉ ATPase (SERCA2a) activity is decreased, resulting in abnormal calcium handling. The disturbances in calcium metabolism have been shown to contribute significantly to the contractile dysfunction observed in heart failure. We investigated whether increasing SERCA2a expression can improve ventricular function in an animal model of heart failure obtained by creating ascending aortic constriction in rats. After 19 -23 wk of banding during the transition from compensated hypertrophy to heart failure (documented by >25% decrease in fractional shortening), rats were randomized to receive either an adenovirus carrying the SERCA2a gene (Ad.SERCA2a, n ‫؍‬ 13) or ␤-galactosidase (Ad.␤gal, n ‫؍‬ 14) by using a catheter-based technique. The failing hearts infected with Ad.␤gal were characterized by a significant decrease in SERCA2a expression and a decrease in SERCA2a activity compared with nonfailing sham-operated rats (n ‫؍‬ 11). In addition, these failing hearts had reduced left-ventricular systolic pressure, maximal rate of left-ventricular pressure rise and decline (؉dP͞dt, ؊dP͞dt), and rate of isovolumic relaxation (). Overexpression of SERCA2a restored both SERCA2a expression and ATPase activity to nonfailing levels. Furthermore, rats infected with Ad.SERCA2a had significant improvement in left-ventricular systolic pressure, ؉dP͞dt, ؊dP͞dt, and rate of isovolumic relaxation () normalizing them back to levels comparable to sham-operated rats. In this study, we show that in an animal model of heart failure where SERCA2a protein levels and activity are decreased and severe contractile dysfunction is present, overexpression of SERCA2a in vivo restores both systolic and diastolic function to normal levels. B oth contraction and relaxation abnormalities at the cardiac myocyte level have been identified in human and animal models of heart failure (1, 2). Trabeculae and isolated cardiac cells from failing hearts have characteristic functional abnormalities, which include an increase in diastolic Ca 2ϩ , an increase in the time course of Ca 2ϩ transient, and a decrease in sarcoplasmic reticulum (SR) Ca 2ϩ release (1-3). These abnormalities are especially accentuated at high frequencies of stimulation leading to the characteristic negative force-frequency relationship in failing myocardium (4).Because the SR plays a central role in controlling Ca 2ϩ movements in myocardial cells during excitation-contraction coupling, a large number of studies have been performed examining the expression and function of the SR Ca 2ϩ ATPase (SERCA2a)(5-9). SERCA2a activity and SR Ca 2ϩ uptake are reduced in failing hearts (9). This reduction in activity is generally, but not invariably, associated with reductions in SERCA2a mRNA and protein. Taken together, these results support the hypothesis that abnormal Ca 2ϩ handling in failing hearts is caused in part by a decrease in SERCA2a activity.To examine the importance of SERCA2a in the development of decompensated heart...

We used a catheter-based technique to achieve generalized cardiac gene transfer in vivo and to alter cardiac function by overexpressing phospholamban (PL) which regulates the activity of the sarcoplasmic reticulum Ca 2؉ ATPase (SERCA2a). By using this approach, rat hearts were transduced in vivo with 5 ؋ 10 9 pfu of recombinant adenoviral vectors carrying cDNA for either PL, ␤-galactosidase (␤-gal), or modified green f luorescent protein (EGFP). Western blot analysis of ventricles obtained from rats transduced by Ad.PL showed a 2.8-fold increase in PL compared with hearts transduced by Ad.␤gal. Two days after infection, rat hearts transduced with Ad.PL had lower peak left ventricular pressure (58.3 ؎ 12.9 mmHg, n ‫؍‬ 8) compared with uninfected hearts (92.5 ؎ 3.5 mmHg, n ‫؍‬ 6) or hearts infected with Ad.␤gal (92.6 ؎ 5.9 mmHg, n ‫؍‬ 6). Both peak rate of pressure rise and pressure fall (؉3, 210 ؎ 298 mmHg͞s, ؊2, 117 ؎ 178 mmHg͞s, n ‫؍‬ 8) were decreased in hearts overexpressing PL compared with uninfected hearts (؉5, 225 ؎ 136 mmHg͞s, ؊3, 805 ؎ 97 mmHg͞s, n ‫؍‬ 6) or hearts infected with Ad.␤gal (؉5, 108 ؎ 167 mmHg͞s, ؊3, 765 ؎ 121 mmHg͞s, n ‫؍‬ 6). The time constant of left ventricular relaxation increased significantly in hearts overexpressing PL (33.4 ؎ 3.2 ms, n ‫؍‬ 8) compared with uninfected hearts (18.5 ؎ 1.0 ms, n ‫؍‬ 6) or hearts infected with Ad.␤gal (20.8 ؎ 2.1 ms, n ‫؍‬ 6). These differences in ventricular function were maintained 7 days after infection. These studies open the prospect of using somatic gene transfer to modulate overall cardiac function in vivo for either experimental or therapeutic applications.The regulation of intracellular calcium is intimately related to the systolic and diastolic function of cardiac cells (1, 2). The sarcoplasmic reticulum (SR), which releases calcium during systole and takes it up during diastole, plays an integral part in controlling the synchronized movement of calcium in myocardial cells. The SR Ca 2ϩ ATPase (SERCA2a) pump regulates the uptake of Ca 2ϩ into the SR during diastole. The function of the SERCA2a pump is regulated in turn by phospholamban (PL) (3). In its unphosphorylated form, PL inhibits the SERCA2a pump whereas in its phosphorylated form, this inhibition is relieved. A decrease in SERCA2a activity has been identified in a number of animal models of heart failure and in human heart failure and an increase in the relative ratio of PL to SERCA2a appears to be an important characteristic of both experimental and human heart failure (3, 4). We have previously modeled such alteration in the PL͞SERCA2a ratio by using adenoviral gene transfer to cardiocytes in vitro. Adenoviral overexpression of PL in vitro recapitulates many of the physiological abnormalities seen in heart failure, including prolonged relaxation and decreased contractile function. In contrast, overexpression of SERCA2a enhances relaxation and contractility of normal cardiomyocytes and rescues myocytes overexpressing PL from their abnormal phenotype (5, 6). Cardiac gene transf...

Abstract-Abnormal calcium cycling, characteristic of experimental and human heart failure, is associated with impaired sarcoplasmic reticulum calcium uptake activity. This reflects decreases in the cAMP-pathway signaling and increases in type 1 phosphatase activity. The increased protein phosphatase 1 activity is partially due to dephosphorylation and inactivation of its inhibitor-1, promoting dephosphorylation of phospholamban and inhibition of the sarcoplasmic reticulum calcium-pump. Indeed, cardiac-specific expression of a constitutively active inhibitor-1 results in selective enhancement of phospholamban phosphorylation and augmented cardiac contractility at the cellular and intact animal levels. Furthermore, the ␤-adrenergic response is enhanced in the transgenic hearts compared with wild types. On aortic constriction, the hypercontractile cardiac function is maintained, hypertrophy is attenuated and there is no decompensation in the transgenics compared with wild-type controls. Notably, acute adenoviral gene delivery of the active inhibitor-1, completely restores function and partially reverses remodeling, including normalization of the hyperactivated p38, in the setting of pre-existing heart failure. Thus, the inhibitor 1 of the type 1 phosphatase may represent an attractive new therapeutic target. Key Words: protein phosphatase 1 Ⅲ protein phosphatase 1 inhibitor 1 Ⅲ heart failure Ⅲ hypertrophy Ⅲ phospholamban Ⅲ gene therapy R eversible protein phosphorylation represents the cellular basis for integration of key signaling pathways, mediating a fine crosstalk between external effector molecules and intracellular events. In the heart, Ca 2ϩ cycling and contractility are controlled by a fine balance of protein kinase and phosphatase activities in response to various second messenger signals. Demands on the heart's pumping action, during fight-or-flight situations, can increase human cardiac output by nearly 5-fold. This is linked to ␤-adrenergic activation of the cAMP dependent protein kinase (PKA). PKA then phosphorylates a set of key regulatory Ca 2ϩ handling proteins that control excitation-contraction coupling cycle, such as phospholamban, the ryanodine receptor, the L-type Ca 2ϩ channel, and troponin I. 1 The protein kinases and their phosphoprotein substrates underlying augmentation of the heart's pumping action have been well characterized. However, similar studies on the protein phosphatases, reversing the increased cardiac contractility, are less well developed. The major Ser/Thr phosphatases [type 1, type 2A, and type 2B (calcineurin)] stem from a common gene family and are highly homologous proteins (40% to 50%) that play critical roles in the control of cardiac contractility and hypertrophy.Overexpression of the catalytic subunit of the protein phosphatase 1 at similar levels observed in human heart failure was associated with dephosphorylation of phospholamban, depressed cardiac function, dilated cardiomyopathy, and premature mortality. 2 Furthermore, PP2A and PP2B (calcineurin) overexpressio...

Abnormal intracellular Ca 2؉ cycling plays an important role in cardiac dysfunction and ventricular arrhythmias in the setting of heart failure and transient cardiac ischemia followed by reperfusion (I͞R). We hypothesized that overexpression of the sarcoplasmic reticulum Ca 2؉ ATPase pump (SERCA2a) may improve both contractile dysfunction and ventricular arrhythmias. Continuous ECG recordings were obtained in 46 conscious rats after adenoviral gene transfer of either SERCA2a or the reporter gene ␤-galactosidase (␤gal) or parvalbumin (PV), as early as 48 h before and 48 h after 30 min ligation of the left anterior descending artery by using an implantable telemetry system. Sham-operated animals were used for comparison for hemodynamic measurements, whereas within-animal baseline was used for electrocardiographic and echocardiographic parameters. All episodes of nonsustained ventricular tachycardia (VT) and ventricular fibrillation (VF) were counted, and their durations were summed by telemetry. I͞R decreased regional cardiac wall thickening as well as the maximal rate of left ventricular pressure rise (؉dP͞dt) and ventricular pressure fall (؊dP͞dt). SERCA2a restored regional wall thickening and ؉dP͞dt and ؊dP͞dt to levels seen preoperatively. Regionalwall motion and anterior-wall thickening were improved in the SERCA2a animals, as assessed by echocardiography and piezoelectric crystals. To assess whether these effects are SERCA2a specific, we overexpressed a skeletal-muscle protein, PV, to examine whether Ca 2؉ buffering alone can mitigate ventricular arrhythmias. During the first hour after I͞R, the rate of nonsustained VT plus VF was 16 ؎ 5 episodes per h (n ‫؍‬ 6) in the Ad.␤gal group, 22 ؎ 6 in the Ad.PV group, and 4 ؎ 2 (n ‫؍‬ 6, P < 0.01) in the Ad.SERCA2a group. The decrease in VT plus VF in the Ad.SERCA2a group was consistent throughout the 48 h of monitoring. These results show that improving intracellular Ca 2؉ handling by overexpression of SERCA2a restores contractile function and reduces ventricular arrhythmias during I͞R.

The relation of the Doppler transmitral flow velocity profile to left ventricular loading conditions and diastolic properties remains poorly described. We studied seven adult mongrel dogs with an open-chest right heart bypass model in which left atrial pressure, representing preload, was varied by controlling blood flow into the pulmonary artery and left ventricular systolic pressure, representing afterload, was controlled independently by pumping blood into or from the femoral arteries. Heart rate was kept constant by crushing the sinus node and pacing the right atrium.Mitral inflow velocity profiles were measured by pulsed-wave Doppler echocardiography at multiple left atrial and left ventricular systolic pressures. In individual dogs, the peak E-wave velocity increased linearly with increasing left atrial V-wave pressure at constant left ventricular systolic pressure and decreased with increasing left ventricular systolic pressure at constant left atrial pressure. Stepwise multiple linear regression analysis of data pooled from all experimental stages in all dogs identified left atrial V-wave pressure, the time constant of relaxation (TL), and left ventricular systolic pressure, in order of decreasing significance, as predictors of the peak E-wave velocity (n = 82, multiple r= 0.87, p<0.OOOl). Multivariate analysis with the same three factors in individual dogs yielded higher r values (mean r= 0.89; range, 0.85-0.97), suggesting the presence of important interdog differences that were not accounted for by these three factors alone. When the values of codeterminant hemodynamic factors were kept within narrower limits, correlations between peak E-wave velocity and left atrial V-wave pressure (n = 35, multiple r= 0.83, p<0.0001), TL (n = 76, multiple r= -0.54, p

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Background-Tissue Doppler imaging (TDI) is a novel echocardiographic method to quantify regional myocardial function. The objective of this study was to assess whether myocardial velocities and strain rate (SR) could be obtained by TDI in mice and whether these indices accurately quantified alterations in left ventricular (LV) systolic function. Methods and Results-TDI was performed in 10 healthy mice to measure endocardial (v endo ) and epicardial systolic velocities and SR. In further experiments, TDI indices were compared with dP/dt max and with sonomicrometer-derived regional velocities, at rest and after administration of dobutamine or esmolol. TDI indices were also studied serially in 8 mice before and 4 and 7 hours after endotoxin challenge.

Localized thrombosis was produced in the left anterior descending (LAD) coronary artery of open chest dogs by constricting a segment so as to produce > 90% stenosis (reducing blood flow to 40±10% of baseline), and placing a thrombus in the segment immediately proximal to the stenosis by inducing endothelial cell injury and instilling a mixture of blood and thrombin.Intravenous infusion of recombinant tissue-type plasminogen activator (rt-PA) at a rate of 15-30 ,g/kg per min for 30 or 60 min in eight dogs induced coronary artery reperfusion within 23±7 min (mean±SD), but reocclusion occurred despite heparin anticoagulation in all but one of these dogs within 7±5 mi.