SERCA2A overexpression decreases the incidence of aftercontractions in adult rabbit ventricular myocytes.

K. Davia, E. Bernobich, H. K. Ranu, F. del Monte, C. M. N. Terracciano, K. T. MacLeod, D. L. Adamson, B. Chaudhri, R. J. Hajjar and S. E. Harding. SERCA2a Overexpression Decreases the Incidence of Aftercontractions in Adult Rabbit Ventricular Myocytes. Journal of Molecular and Cellular Cardiology (2001) 33, 1005-1015. Slow relaxation and poor contractile response to increasing stimulation frequency in failing human heart have been strongly linked to a decrease in the activity of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a). Restoration of SERCA2a levels using gene transfer has beneficial effects on contractile function but, like beta -adrenoceptor stimulation, could potentially produce excess SR Ca(2+), arrhythmias and cell death. We have examined the effects of SERCA2a overexpression in adult rabbit cardiac myocytes, and compared changes in relaxation with those following beta -adrenoceptor stimulation. Myocytes were infected with an adenovirus carrying both SERCA2a and green fluorescent protein (GFP) for positive identification of infected cells. Myocyte survival was significantly enhanced in the infected cultures. There was a reduction in both time-to-peak contraction and time-to-50% relaxation (R50) 48 h after infection. Time-to-90% relaxation (R90) was particularly improved (non-infected 516+/-41 ms, AD.SERCA2a-GFP 230+/-23 ms, n=7 preparations, P<0.001). There was also a decreased incidence of aftercontractions in Ad.SERCA2a-GFP infected myocytes (21+/-5%v 41+/-4% in controls, P<0.01). This contrasts with beta -adrenoceptor stimulation, which reduced R50 but prolonged R90 by 158+/-76 ms (P<0.02, n=16). At higher stimulation frequencies (2-3 Hz) contraction amplitude and SR calcium content were increased and diastolic contracture was reduced following SERCA2a overexpression. Overall, increasing levels of SERCA2a resulted in an improvement in systolic and diastolic function and a reduction in cell death and arrhythmic aftercontractions. SERCA2a overexpression therefore lacks the detrimental effects associated with some other inotropic interventions.

Characterisation of the function of adult guinea-pig ventricular myocytes following co-culture with neonatal rat myocytes.

Adult guinea-pig myocytes were co-cultured with a layer of spontaneously contracting neonatal rat myocytes based on a method described by Weisensee D. (In Vitro Cell Dev Biol 31: 190-195, 1995). Contractile studies were performed on freshly isolated, 24 and 48 h co-cultured adult guinea-pig myocytes to investigate whether alterations in contractile function had occurred. No difference was found between freshly isolated and 24 h co-cultured adult guinea-pig myocytes in terms of sensitivity to calcium, isoprenaline, frequency response and beat duration. After 48 h, the frequency response was depressed (P < 0.02) and the beat was prolonged (P < 0.05) when compared to that of freshly isolated myocytes. In the presence of the SR Ca2+ ATPase inhibitor, thapsigargin, the beat was significantly prolonged (P = 0.003) in 24 h co-cultured myocytes but not in freshly isolated myocytes. These findings show that adult guinea-pig myocytes can be maintained in co-culture with neonatal rat myocytes with little change in contractile function for 24 h but after this time contractile function begins to deteriorate.

Functional alterations in adult rat myocytes after overexpression of phospholamban with use of adenovirus.

An increased phospholamban (PLB)-to-sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) ratio has been suggested to contribute to the slowing of relaxation in failing human ventricle. We have used an adenoviral vector carrying the sequence for PLB to increase this ratio in isolated adult rat ventricular myocytes, and we have examined the functional consequences. With use of adenoviral vectors, the PLB content of adult rat myocytes was increased 2.73-fold, with SERCA2a levels unchanged. Maximum contraction amplitude of PLB-overexpressing myocytes was decreased to 6.9 +/- 0.3% shortening compared with 11.2 +/- 0.8% for 24-h controls (Con; P < 0.001, 5 preparations, 103 myocytes). Maximum rates of shortening and relengthening were also significantly decreased. Ca(2+) transient amplitudes were slightly depressed, and time to 50% decay of the transients was significantly increased: 237 +/- 18 (n = 14 myocytes) and 432 +/- 32 ms in Con and PLB (n = 15) myocytes, respectively (P < 0.001). The amount of Ca(2+) in the sarcoplasmic reticulum stores was reduced by 21% (P < 0.05). Relaxation was significantly slower in PLB than in Con myocytes when the Na(+)/Ca(2+) exchanger was blocked but not when sarcoplasmic reticulum Ca(2+) uptake was inhibited. Adenovirus infection with Ad.RSV.PLB was therefore able to produce functional changes in adult cardiac myocytes within 24 h, consistent with overexpression of PLB and similar to those seen in failing human heart.

Post-rest contraction amplitude in myocytes from failing human ventricle.

It has been reported that the balance between the two main Ca2+ removal systems in the cardiac cells, the sarcoplasmic reticulum (SR) and Na+/Ca2+ exchanger, is altered in failing human heart. We have studied post-rest contraction behaviour as a non-invasive probe of the amount of Ca2+ stored in the SR in myocytes from failing and non-failing human ventricle. The first beat following a rest interval, as a percentage of the preceding steady state (B1/SS), was larger and more variable in cells from failing heart, indicating some accumulation of Ca2+ in the SR during rest. This could be mimicked by treatment of myocytes with digoxigenin, a compound which increases intracellular Na+, suggesting that alterations in the Na+ balance of the cell might contribute to the effect. Isoprenaline, which stimulates Ca2+ uptake by the SR while the myocyte is beating, prevented SR Ca2+ accumulation during rest in susceptible myocytes. We hypothesize that loss of SR function in the failing heart is partially compensated for by increased Ca2+ extrusion via the Na+/Ca2+ exchange in the contracting myocyte, leading to increased intracellular Na+ during activity. This Na+ is lost at rest, predisposing the cells to accumulate Ca2+ in the SR. Experiments to test this hypothesis are proposed.

From overload to failure: what happens inside the myocyte.

To determine whether there is a defect in the surviving muscle cells of the failing human heart, studies have been performed on individual myocytes isolated from normal and failing human myocardium. Myocytes from the failing ventricle contract and relax more slowly, and have a reduced contraction amplitude at physiological (but not low) stimulation frequencies. Slow relaxation is seen irrespective of the aetiology of the heart disease studied, and is more pronounced in myocytes from hypertrophied ventricles. Myocytes from hypertrophied ventricles are larger than normal, but the relaxation deficit is independent of cell size. Beta-adrenoceptor desensitization is evident in myocytes and it varies according to the severity of disease and with the age of the patient. Action potentials are longer in myocytes from failing human heart, probably because of an alteration in K+ current density. Many of the functional changes identified in failing human myocardium are seen at the level of the single cardiac myocyte, which implies that pharmacological or genetic manipulation of surviving cells is a logical therapeutic strategy.

Effects of inhibition of sarcoplasmic reticulum calcium uptake on contraction in myocytes isolated from failing human ventricle.

OBJECTIVES: There has been debate regarding the level of sarcoplasmic reticulum (SR) Ca2+ ATPase protein in heart failure. We have used the SR Ca2+ ATPase inhibitor thapsigargin to investigate the functional contribution of this uptake system to contraction and relaxation in myocytes from failing and non-failing human ventricle. METHODS: Myocytes were isolated from 28 failing and 18 non-failing ventricles and stimulated at 0.2 Hz, 32 degrees C in Krebs-Henseleit solution. Contraction amplitude and speed were compared before and after treatment with 1 mumol/l thapsigargin for 20 min to deplete SR Ca2+ stores. RESULTS: Initial beat duration was longer in myocytes from failing hearts. Addition of thapsigargin significantly prolonged the beat in myocytes from both groups, but the increase was greater in non-failing hearts (beat duration increased by 0.79 +/- 0.12 s in myocytes from non-failing hearts compared with 0.37 +/- 0.12 s in those from failing, P < 0.02). The contraction amplitude increased at high stimulation frequencies in myocytes from non-failing hearts (from 2.6% shortening at 0.1 Hz to 4.6% at 1 Hz, P < 0.001, n = 9), but not in those from failing hearts (1.8% at 0.1 Hz compared with 1.7% at 1 Hz, n = 5). Thapsigargin abolished the positive staircase in the non-failing, but had no effect in the failing group. Contraction amplitude following a rest interval was significantly depressed relative to steady-state levels in myocytes from the non-failing hearts (44.8 +/- 10.3% at 3 min), but not in failing (102 +/- 18%, P < 0.01 compared to non-failing at 3 min). Following thapsigargin treatment, there were no longer significant differences between failing and non-failing myocytes in the time course of the beat, frequency response or post-rest behaviour. CONCLUSION: The differences between myocytes from failing and non-failing hearts were reduced by inhibition of SR function. These results are consistent with the hypothesis that the initial differences had been due to decreased SR Ca2+ uptake.

Alterations in beta-adrenoceptor mechanisms in the aging heart. Relationship with heart failure.

In chronic heart failure substantial and characteristic changes occur in the function of the adrenergic nervous system. Studies in isolated left ventricular muscle and in single cardiomyocytes from experimental models of aging and, recently, from humans show an age-related reduced contractile response to beta-adrenoceptor stimulation. "beta-adrenoceptor desensitization" is thought to be a general and common mechanism to explain the age- and heart failure-related decrease in beta-adrenoceptor response. The aim of this review is to compare alterations in beta-adrenoceptor mechanisms in physiological cardiovascular aging and chronic heart failure. From an analysis of the overall data on the role of aging in beta-adrenoceptor regulation in human and animal hearts, it is possible to conclude that the reduced response to beta-agonists is common to all species and all cardiac tissues. Moreover, the age-related changes are limited to beta-adrenoceptor-G-protein (s)-adenylyl cyclase system abnormalities, while the type and level of abnormalities change with species and tissues. The modifications shown in the aging heart are not very different from some observed in heart failure. In particular, both in aged and failing hearts we may see that the decrease in beta-adrenoceptor responsiveness is related to changes in G-protein function.

Tachycardia-induced failure alters contractile properties of canine ventricular myocytes.

OBJECTIVE: Rapid cardiac pacing has been used as a model for experimentally-induced cardiomyopathy. However, its relevance to human heart failure is not clear at present because little is known about changes in size and function of ventricular myocytes. We have therefore studied the responses to graded increases in frequency and calcium in canine ventricular myocytes from failing hearts. The aim of our study was to evaluate the resemblance between canine pacing-induced and human end-stage heart failure. METHODS: Myocytes were isolated from the left ventricular wall of dogs that were in heart failure after 6 weeks of pacing at 250 beats/min. Cell shortening was measured by edge detection. RESULTS: Clinical signs of failure included dyspnea, ascites, and heart dilatation; the hemodynamic parameters were: LVdP/dtmax 1613 +/- 149 vs. 4713 +/- 304 mmHg/s in 6 control dogs; LVEDP 17.2 +/- 4.4 vs 5.6 +/- 1.1 mmHg; LV volume 60.5 +/- 6.2 vs. 30-35 ml. Myocytes from failing hearts were longer and thinner than those from controls (from factor: 0.40 +/- 0.01 vs. 0.47 +/- 0.01, P < 0.001, > 30 cells/heart). With 6 mM Ca2+ and at 0.5 Hz, contraction amplitude was significantly attenuated in myocytes from failing hearts: 6.6 +/- 0.9% cell shortening vs. 10.0 +/- 0.8% in controls (P < 0.05). This deficit was exacerbated at higher stimulation rates. Time-to-peak contraction and time-to-50% relaxation were not altered. There was no difference in sensitivity to thapsigargin. CONCLUSION: As with cells from human failing hearts, contraction amplitude showed rate-dependent depression in this animal model, whereas features like slowing of contraction and relaxation and reduced sensitivity to thapsigargin, were not reproduced.

Phosphodiesterase inhibition and Ca2+ sensitization.

Inhibitors of phosphodiesterase type III (PDE III) enhance cardiac contractile force by elevating the intracellular calcium concentration [Ca2+]i by impairing cAMP degradation thus increasing cAMP levels. The drugs are more effective in healthy than in failing hearts since basal cAMP production is diminished in the latter. However, long term treatment with PDE-III inhibitors does not appear to be beneficial due to increased risk of potentially lethal arrhythmias caused by augmentation of [Ca2+]i[1). This risk should be absent in Ca2+ sensitizers. Recently, thiadiazinone derivatives have been synthetized in which the potency for Ca2+ sensitization is many-fold larger than the potency for PDE-III inhibition. The Ca(2+)-sensitizing action resides in the [+]-enantiomers, while the [-]-enantiomers show weak PDE-III inhibition. In the enantiomer pair [+]-EMD 60263 and [-]-EMD 60264, only the former concentration-dependently increased force of contraction in isolated cardiac preparations and myocytes. In the Langendorff-perfused guinea-pig heart, force was reversibly increased, whereas [-]-EMD 60264 even produced a negative inotropic response despite of its PDE inhibitory activity. Heart rate, however, was reduced by both enantiomers. Perfusion pressure remained unaffected. The effects were fully reversible upon wash-out of the enantiomers. [+]-EMD 60263 also enhanced cell shortening of human myocytes from both normal and failing hearts. In contrast to the opposite effects on contractility, both enantiomers prolong the action potential duration by blocking the rapidly activating component of the delayed rectifier K+ current. Thus they also possess class III antiarrhythmic activity. The therapeutic potential of these agents has yet to be assessed in clinical studies.

Reduced contraction and altered frequency response of isolated ventricular myocytes from patients with heart failure.

BACKGROUND: Previous work has failed to demonstrate reduced maximal contraction of isolated ventricular myocytes from failing human hearts compared with nonfailing control hearts. The effect of alterations in stimulation frequency and temperature on the contraction of isolated ventricular myocytes has been investigated. Left ventricular myocytes were isolated from the hearts of patients with severe heart failure undergoing heart transplantation and compared with myocytes isolated from myocardial biopsies from patients with coronary disease but preserved left ventricular systolic function or from myocytes from rejected donor hearts. METHODS AND RESULTS: Myocytes were exposed to either a maximally activating level of extracellular calcium at 37 degrees C or to 2 mmol/L calcium at 32 degrees C. There was no significant difference in the contraction amplitude between myocytes from failing and nonfailing hearts at 0.2 Hz. With increasing stimulation frequency, there was a reduction in contraction amplitude in cells from failing hearts relative to control hearts in both maximal calcium from 0.33 Hz (4.5% versus 6.6%) to 1.4 Hz (3.9% versus 8.8%) (ANCOVA, P < .001) and at 2 mmol/L calcium from 0.50 Hz (2.3% versus 3.5%) to 1.4 Hz (1.8% versus 3.9%) (ANCOVA, P < .001). The time to peak contraction and the times to 50% and 90% relaxation were prolonged in myocytes from failing hearts at stimulation rate of 0.2 Hz (P < .01), but only the time to 50% relaxation was prolonged at 1.0 Hz (P < .05). CONCLUSIONS: Reduced contraction, slowed relaxation, and impaired frequency response occurring at the level of the individual ventricular myocyte can be demonstrated in human heart failure. This demonstrates that disruption of myocyte function can contribute to both the systolic and the diastolic abnormalities that occur in the failing human heart.