SERCA2a Overexpression Decreases the Incidence of Aftercontractions in Adult Rabbit Ventricular Myocytes (original) (raw)
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Cardiovascular Research, 2003
Objective: The activity of sarcoplasmic reticulum Ca -ATPase (SERCA) is reduced in the failing myocardium. Therefore, transfer of SERCA2a cDNA is considered as a therapeutical approach. The aim of this study was analysis of the long-term effect of SERCA2a overexpression in normal as well as pressure overload challenged myocardium of transgenic rats. Methods: Independent transgenic rat lines were established expressing the rat SERCA2a cDNA specifically in the myocardium resulting in increased SERCA2a protein levels by 30-70%. Simultaneous measurements of isometric contraction and calcium transients were carried out in right ventricular papillary muscle preparations. Hemodynamic parameters were measured in hearts of unchallenged rats as well as 10 weeks after pressure overload induced by abdominal aortic banding. Results: Analysis of calcium handling and contractile parameters in isolated right ventricular papillary muscles revealed significant shortening of intracellular calcium transients and half maximal relaxation times (RT ). Assessing 50 myocardial contractility in working heart preparations, both transgenic rat lines revealed elevated left ventricular pressure, improved systolic and diastolic parameters, attenuated negative force-frequency relation, and a dose-dependent b-adrenergic effect. Aortic banding resulted in reduction of left ventricular pressure and worsening of contraction and relaxation parameters with no differences in mortality in both transgenic (1dP/dt 3084696 vs. 39386250 mmHg / s; RT 47.061.2 vs. 36.761.4 ms) and wild-type rats (1dP/dt 2695686 vs. 50 32976122 mmHg / s; RT 53.061.6 vs. 44.161.4). SERCA2a overexpressing hearts revealed improved hemodynamic parameters 50 compared to wild-type controls. Acceleration of isovolumetric relaxation characterized by the index Tau was directly correlated to SERCA2a protein concentrations. Conclusion: Overexpression of SERCA2a protein results in a positive inotropic effect under baseline conditions remaining preserved under pressure overload without affecting mortality. Therefore therapeutic transfer of SERCA2a may become a potential approach for gene therapy of congestive heart failure. Moreover, transgenic SERCA2a rats will be useful for studies of long-term SERCA2a overexpression in further cardiovascular disease models.
Journal of molecular and …, 2006
Abnormal Ca 2+ cycling in the failing heart might be corrected by enhancing the activity of the cardiac Ca 2+ pump, the sarco(endo)plasmic reticulum Ca 2+ -ATPase 2a (SERCA2a) isoform. This can be obtained by increasing the pump's affinity for Ca 2+ by suppressing phospholamban (PLB) activity, the in vivo inhibitor of SERCA2a. In SKO mice, gene-targeted replacement of SERCA2a by SERCA2b, a pump with a higher Ca 2+ affinity, results in cardiac hypertrophy and dysfunction. The stronger PLB inhibition on cardiac morphology and performance observed in SKO was investigated here in DKO mice, which were obtained by crossing SKO with PLB −/− mice. The affinity for Ca 2+ of SERCA2 was found to be further increased in these DKO mice. Relative to wild-type and SKO mice, DKO mice were much less spontaneously active and showed a reduced life span. The DKO mice also displayed a severe cardiac phenotype characterized by a more pronounced concentric hypertrophy, diastolic dysfunction and increased ventricular stiffness. Strikingly, beta-adrenergic or forced exercise stress induced acute heart failure and death in DKO mice. Therefore, the increased PLB inhibition represents a compensation for the imposed high Ca 2+ -affinity of SERCA2b in the SKO heart. Limiting SERCA2's affinity for Ca 2+ is physiologically important for normal cardiac function. An improved Ca 2+ transport in the sarcoplasmic reticulum may correct Ca 2+ mishandling in heart failure, but a SERCA pump with a much higher Ca 2+ affinity may be detrimental.
Effects of sarcoplasmic reticulum Ca2+-ATPase overexpression in postinfarction rat myocytes
Journal of Applied Physiology, 2005
2+ concentration ([Ca 2+ ] i ) homeostasis, decreased sarcoplasmic reticulum (SR) Ca 2+ -ATPase (SERCA2) expression and activity, but SR Ca 2+ leak was unchanged. In the present study, we investigated whether SERCA2 overexpression in MI myocytes would restore contraction and [Ca 2+ ] i transients to normal. Compared to sham-operated hearts, 3 week MI hearts exhibited significantly higher left ventricular (LV) end-diastolic and endsystolic volumes, but lower fractional shortening and ejection fraction as measured by Mmode echocardiography. Seventy-two hours after adenovirus-mediated gene transfer, SERCA2 overexpression in 3 week MI myocytes did not affect Na + /Ca 2+ exchanger expression, but restored the depressed SERCA2 levels towards those measured in sham myocytes. In addition, the reduced SR Ca 2+ uptake in MI myocytes was improved to normal levels by SERCA2 overexpression. At extracellular Ca 2+ concentration ([Ca 2+ ] o ) of 5 mM, the subnormal contraction and [Ca 2+ ] i transient amplitudes in MI myocytes (compared with sham myocytes) were restored to normal by SERCA2 overexpression. However, at 0.6mM [Ca 2+ ] o , the supernormal contraction and [Ca 2+ ] i transient amplitudes in MI myocytes (compared with sham myocytes) were exacerbated by SERCA2 overexpression. We conclude that SERCA2 overexpression was only partially effective in ameliorating contraction and [Ca 2+ ] i transient abnormalities in our rat model of ischemic cardiomyopathy. We suggest that other Ca 2+ transport pathways, e.g., Na + /Ca 2+ exchanger, may also play an important role in contractile and [Ca 2+ ] i homeostatic abnormalities in MI myocytes. FINAL ACCEPTED VERSION
Journal of Biological Chemistry, 2000
The sarcoplasmic reticulum calcium ATPase SERCA2b is an alternate isoform encoded by the SERCA2 gene. SERCA2b is expressed ubiquitously and has a higher Ca 2؉ affinity compared with SERCA2a. We made transgenic mice that overexpress the rat SERCA2b cDNA in the heart. SERCA2b mRNA level was approximately ϳ20-fold higher than endogenous SERCA2b mRNA in transgenic hearts. SERCA2b protein was increased 8-10-fold in the heart, whereas SERCA2a mRNA/protein level remained unchanged. Confocal microscopy showed that SERCA2b is localized preferentially around the T-tubules of the SR, whereas SERCA2a isoform is distributed both transversely and longitudinally in the SR membrane. Calciumdependent calcium uptake measurements showed that the maximal velocity of Ca 2؉ uptake was not changed, but the apparent pump affinity for Ca 2؉ (K 0.5) was increased in SERCA2b transgenic mice (0.199 ؎ 0.011 M) compared with wild-type control mice (0.269 ؎ 0.012 M, p < 0.01). Work-performing heart preparations showed that SERCA2b transgenic hearts had a higher rates of contraction and relaxation, shorter time to peak pressure and half-time for relaxation than wild-type hearts. These data show that SERCA2b is associated in a subcompartment within the sarcoplasmic reticulum of cardiac myocytes. Overexpression of SERCA2b leads to an increase in SR calcium transport function and increased cardiac contractility, suggesting that SERCA2b plays a highly specialized role in regulating the beat-to-beat contraction of the heart.
Cardiovascular research, 2005
Heart failure is associated with reduced function of sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) but increased function of sarcolemmal Na+/Ca2+ exchanger (NCX), leading to decreased SR Ca2+ content and loss of frequency-potentiation of contractile force. We reported that SERCA2a-overexpression in transgenic rat hearts (TG) results in improved contractility. However, it was not clear whether TG have improved contractility due to frequency-dependent improved SR Ca2+ handling. Therefore, we characterized TG (n=35) vs. wild-type (WT) control rats (n=39) under physiological conditions (37 degrees C, stimulation rate <8 Hz). Twitch force, intracellular Ca2+ transients ([Ca2+]i), and SR Ca2+ content were measured in isolated muscles. The contribution of transsarcolemmal Ca2+ influx (I(Ca)) through L-type Ca2+ channels (LTCC) and reverse mode NCX (I(Na/Ca)) to Ca2+ cycling were studied in isolated myocytes. With increasing frequency, force increased in TG muscles by 168+/-35% (8 Hz...
Enhanced L-type calcium currents in cardiomyocytes from transgenic rats overexpressing SERCA2a
Experimental and Clinical Cardiology, 2010
Previous research reported that transgenic rats overexpressing the sarco(endo)plasmic reticulum Ca(2+)-ATPase SERCA2a exhibit improved contractile function of the myocardium. Furthermore, impaired Ca(2+) uptake and reduced relaxation rates in rats with diabetic cardiomyopathy were partially rescued by transgenic expression of SERCA2a in the heart. To explore whether enhanced Ca(2+) cycling in the cardiomyocytes of SERCA2a transgenic rats is associated with changes in L-type Ca(2+) (I(Ca-L)) currents. The patch-clamp technique was used to measure whole-cell currents in cardiomyocytes from transgenic rats overexpressing SERCA2a and from wild-type (nontransgenic) animals. The amplitudes of I(Ca-L) currents at depolarizing pulses ranging from -45 mV to 0 mV (350 ms duration, 1 Hz) were significantly higher in cardiomyocytes of SERCA2a transgenic rats than in nontransgenic rats (1985±48 pA [n=32] versus 1612±55 pA [n=28], respectively). The inactivation kinetics of I(Ca-L) showed subtle ...