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The cardiac SERCA2 Ca(2+) pump is critical for maintaining normal Ca(2+) handling in the heart. Reduced SERCA2a content and blunted Ca(2+) reuptake are frequently observed in failing hearts and evidence implicates poor cardiac Ca(2+) handling in the progression of heart failure. To gain insight into mechanism we investigated a novel genetic mouse model of inducible severe and progressive SERCA2 deficiency (inducible Serca2 knockout, SERCA2 KO). These mice eventually die from overt heart failure 7-10 weeks after knockout but as yet there have been no reports on intrinsic mechanical performance at the isolated whole heart organ level. Thus we studied whole-organ ex vivo function of hearts isolated from SERCA2 KO mice at one and four weeks post-knockout in adult animals. We found that isolated KO heart function was only modestly impaired one week post-knockout, when SERCA2a protein was 32% of normal. At four weeks post-knockout, function was severely impaired with near non-detectable levels of SERCA2. During perfusion with 10 mM caffeine, LV developed pressures were similar between 4-week KO and control hearts, and end-diastolic pressures were lower in KO. When hearts were subjected to ischemia-reperfusion injury, recovery was not different between control and KO hearts at either one or four weeks post-knockout. Our findings indicate that ex vivo function of isolated SERCA2 KO hearts is severely impaired long before symptoms appear in vivo, suggesting that physiologically relevant heart function in vivo can be sustained for weeks in the absence of robust SR Ca(2+) flux.

In human heart failure (HF), reduced cardiac function has, at least partly, been ascribed to altered calcium homeostasis in cardiomyocytes. The effects of the calcium sensitizer levosimendan on diastolic dysfunction caused by reduced removal of calcium from cytosol in early diastole are not well known. In this study, we investigated the effect of long-term levosimendan treatment in a murine model of HF where the sarco(endo)plasmatic reticulum ATPase (Serca) gene is specifically disrupted in the cardiomyocytes, leading to reduced removal of cytosolic calcium. After induction of Serca2 gene disruption, these mice develop marked diastolic dysfunction as well as impaired contractility. SERCA2 knockout (SERCA2KO) mice were treated with levosimendan or vehicle from the time of KO induction. At the 7-wk end point, cardiac function was assessed by echocardiography and pressure measurements. Vehicle-treated SERCA2KO mice showed significantly diminished left-ventricular (LV) contractility, as shown by decreased ejection fraction, stroke volume, and cardiac output. LV pressure measurements revealed a marked increase in the time constant (τ) of isovolumetric pressure decay, showing impaired relaxation. Levosimendan treatment significantly improved all three systolic parameters. Moreover, a significant reduction in τ toward normalization indicated improved relaxation. Gene-expression analysis, however, revealed an increase in genes related to production of the ECM in animals treated with levosimendan. In conclusion, long-term levosimendan treatment improves both contractility and relaxation in a heart-failure model with marked diastolic dysfunction due to reduced calcium transients. However, altered gene expression related to fibrosis was observed.

Cardiomyocyte contraction and relaxation are controlled by Ca(2+) handling, which can be regulated to meet demand. Indeed, major reduction in sarcoplasmic reticulum (SR) function in mice with Serca2 knockout (KO) is compensated by enhanced plasmalemmal Ca(2+) fluxes. Here we investigate whether altered Ca(2+) fluxes are facilitated by reorganization of cardiomyocyte ultrastructure. Hearts were fixed for electron microscopy and enzymatically dissociated for confocal microscopy and electrophysiology. SR relative surface area and volume densities were reduced by 63% and 76%, indicating marked loss and collapse of the free SR in KO. Although overall cardiomyocyte dimensions were unaltered, total surface area was increased. This resulted from increased T-tubule density, as revealed by confocal images. Fourier analysis indicated a maintained organization of transverse T-tubules but an increased presence of longitudinal T-tubules. This demonstrates a remarkable plasticity of the tubular system in the adult myocardium. Immunocytochemical data showed that the newly grown longitudinal T-tubules contained Na(+)/Ca(2+)-exchanger proximal to ryanodine receptors in the SR but did not contain Ca(2+)-channels. Ca(2+) measurements demonstrated a switch from SR-driven to Ca(2+) influx-driven Ca(2+) transients in KO. Still, SR Ca(2+) release constituted 20% of the Ca(2+) transient in KO. Mathematical modeling suggested that Ca(2+) influx via Na(+)/Ca(2+)-exchange in longitudinal T-tubules triggers release from apposing ryanodine receptors in KO, partially compensating for reduced SERCA by allowing for local Ca(2+) release near the myofilaments. T-tubule proliferation occurs without loss of the original ordered transverse orientation and thus constitutes the basis for compensation of the declining SR function without structural disarrangement.

Sarcoplasmic reticulum Ca(2+) ATPases (SERCAs) play a major role in muscle contractility by pumping Ca(2+) from the cytosol into the sarcoplasmic reticulum (SR) Ca(2+) store, allowing muscle relaxation and refilling of the SR with releasable Ca(2+). Decreased SERCA function has been shown to result in impaired muscle function and disease in human and animal models. In this study, we present a new mouse model with targeted disruption of the Serca2 gene in skeletal muscle (skKO) to investigate the functional consequences of reduced SERCA2 expression in skeletal muscle. SkKO mice were viable and basic muscle structure was intact. SERCA2 abundance was reduced in multiple muscles, and by as much as 95% in soleus muscle, having the highest content of slow-twitch fibres (40%). The Ca(2+) uptake rate was significantly reduced in SR vesicles in total homogenates. We did not find any compensatory increase in SERCA1 or SERCA3 abundance, or altered expression of several other Ca(2+)-handling proteins. Ultrastructural analysis revealed generally well-preserved muscle morphology, but a reduced volume of the longitudinal SR. In contracting soleus muscle in vitro preparations, skKO muscles were able to fully relax, but with a significantly slowed relaxation time compared to controls. Surprisingly, the maximal force and contraction rate were preserved, suggesting that skKO slow-twitch fibres may be able to contribute to the total muscle force despite loss of SERCA2 protein. Thus it is possible that SERCA-independent mechanisms can contribute to muscle contractile function.

Reduced sarco(endo)plasmic reticulum (SR) Ca(2+) ATPase (SERCA2) contributes to the impaired cardiomyocyte Ca(2+) homeostasis observed in heart failure. We hypothesized that a reduction in SERCA2 also elicits myocardial ER/SR stress responses, including unfolded protein responses (UPR) and cardiomyocyte apoptosis, which may additionally contribute to the pathophysiology of this condition. Left ventricular myocardium from mice with cardiomyocyte-specific tamoxifen-inducible disruption of Serca2 (SERCA2 KO) was compared with aged-matched controls. In SERCA2 KO hearts, SERCA2 protein levels were markedly reduced to 2% of control values at 7 weeks following tamoxifen treatment. Serca2 disruption caused increased abundance of the ER stress-associated proteins CRT, GRP78, PERK, and eIF2α and increased phosphorylation of PERK and eIF2α, indicating UPR induction. Pro-apoptotic signaling was also activated in SERCA2 KO, as the abundance of CHOP, caspase 12, and Bax was increased. Indeed, TUNEL staining revealed an increased fraction of cardiomyocytes undergoing apoptosis in SERCA2 KO. ER-Tracker staining additionally revealed altered ER structure. These findings indicate that reduction in SERCA2 protein abundance is associated with marked ER/SR stress in cardiomyocytes, which induces UPR, apoptosis, and ER/SR structural alterations. This suggests that reduced SERCA2 abundance or function may contribute to the phenotype of heart failure also through induction of ER/SR stress responses., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

We describe a simulation study of Ca²(+) dynamics in mice with cardiomyocyte-specific conditional excision of the sarco(endo)plasmic reticulum calcium ATPase (SERCA) gene, using an experimental data-driven biophysically-based modeling framework. Previously, we reported a moderately impaired heart function measured in mice at 4 weeks after SERCA2 gene deletion (knockout (KO)), along with a >95% reduction in the level of SERCA2 protein. We also reported enhanced Ca²(+) flux through the L-type Ca²(+) channels and the Na(+)/Ca²(+) exchanger in ventricular myocytes isolated from these mice, compared to the control Serca2(flox/flox) mice (flox-flox (FF)). In the current study, a mathematical model-based analysis was applied to enable further quantitative investigation into changes in the Ca²(+) handling mechanisms in these KO cardiomyocytes. Model parameterization based on a wide range of experimental measurements showed a 67% reduction in SERCA activity and an over threefold increase in the activity of the Na(+)/Ca²(+) exchanger. The FF and KO models were then validated against experimentally measured [Ca²(+)](i) transients and experimentally estimated sarco(endo)plasmic reticulum (SR) function. Simulation results were in quantitative agreement with experimental measurements, confirming that sustained [Ca²(+)](i) transients could be maintained in the KO cardiomyocytes despite severely impaired SERCA function. In silico analysis shows that diastolic [Ca²(+)](i) rises sharply with progressive reductions in SERCA activity at physiologically relevant pacing frequencies. Furthermore, an analysis of the roles of the compensatory mechanisms revealed that the major combined effect of the compensatory mechanisms is to lower diastolic [Ca²(+)](i). Finally, by using a comprehensive sensitivity analysis of the role of all cellular calcium handling mechanisms, we show that the combination of upregulation of the Na(+)/Ca²(+) exchanger and increased L-type Ca²(+) current is the most effective means to maintain diastolic and systolic calcium levels after loss of SERCA function., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

In the heart, function of the sarco(endo)plasmic Ca(2+)-ATPase (SERCA2) is closely linked to contractility, cardiac function, and aerobic fitness. SERCA2 function can be increased by high-intensity interval training, whereas reduced SERCA2 abundance is associated with impaired cardiac function. The working hypothesis was, therefore, that exercise training before cardiomyocyte-specific disruption of the Serca2 gene would delay the onset of cardiac dysfunction in mice. Before Serca2 gene disruption by tamoxifen, untreated SERCA2 knockout mice (Serca2(flox/flox) Tg-αMHC-MerCreMer; S2KO), and SERCA2 FF control mice (Serca2(flox/flox), S2FF) were exercise trained by high-intensity interval treadmill running for 6 wk. Both genotypes responded to training, with comparable increases in maximal oxygen uptake (Vo(2max); 17%), left ventricle weight (15%), and maximal running speed (40%). After exercise training, cardiac-specific Serca2 gene disruption was induced in both exercise trained and sedentary S2KO mice. In trained S2KO, cardiac function decreased less rapidly than in sedentary S2KO. Vo(2max) remained higher in trained S2KO the first 15 days after gene disruption. Six weeks after Serca2 disruption, cardiac output was higher in trained compared with sedentary S2KO mice. An exercise-training program attenuates the decline in cardiac performance induced by acute cardiac Serca2 gene disruption, indicating that mechanisms other than SERCA2 contribute to the favorable effect of exercise training.

Several lines of evidence indicate that the sarco(endo)plasmic reticulum ATPase type 2 (SERCA2) is essential for maintaining myocardial calcium handling and cardiac pump function. Hence, a reduction in SERCA2 abundance is expected to reduce work performance and maximal oxygen uptake (VO2max) and to limit the response to exercise training. To test this hypothesis, we compared VO2max and exercise capacity in mice with cardiac disruption of Serca2 (SERCA2 KO) with control mice (SERCA2 FF). We also determined whether the effects on VO2max and exercise capacity could be modified by high-intensity aerobic exercise training. Treadmill running at 85-90% of VO2max started 2 wk after Serca2 gene disruption and continued for 4 wk. VO2max and maximal running speed were measured weekly in a metabolic chamber. Cardiac function was assessed by echocardiography during light anesthesia. In sedentary SERCA2 KO mice, the aerobic capacity was reduced by 50% and running speed by 28%, whereas trained SERCA2 KO mice were able to maintain maximal running speed despite a 36% decrease in VO2max. In SERCA2 FF mice, both VO2max and maximal running speed increased by training, while no changes occurred in the sedentary group. Left ventricle dimensions remained unchanged by training in both genotypes. In contrast, training induced right ventricle hypertrophy in SERCA2 KO mice. In conclusion, the SERCA2 protein is essential for sustaining cardiac pump function and exercise capacity. Nevertheless, SERCA2 KO mice were able to maintain maximal running speed in response to exercise training despite a large decrease in VO2max.

Aims: To describe the overall role of reduced sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) for Ca(2+) wave development., Methods and Results: SERCA2 knockout [Serca2(flox/flox) Tg(alphaMHC-MerCreMer); KO] mice allowing inducible cardiomyocyte-specific disruption of the Serca2 gene in adult mice were compared with Serca(flox/flox) (FF) control mice. Six days after Serca2 gene disruption, SERCA2 protein abundance was reduced by 53% in KO compared with FF, whereas SERCA2 activity in field-stimulated, Fluo-5F AM-loaded cells was reduced by 42%. Baseline Ca(2+) content of the sarcoplasmic reticulum (SR) and Ca(2+) transient amplitude and rate constant of decay measured in whole-cell voltage-clamped cells were decreased in KO to 75, 81, and 69% of FF values. Ca(2+) waves developed in only 31% of KO cardiomyocytes compared with 57% of FF when external Ca(2+) was raised (10 mM), although SR Ca(2+) content needed for waves to develop was 79% of FF values. In addition, waves propagated at a 15% lower velocity in KO cells. Ventricular extrasystoles (VES) occurred with lower frequency in SERCA2 KO mice (KO: 3 +/- 1 VES/h vs. FF: 8 +/- 1 VES/h) (P < 0.05 for all results)., Conclusion: Reduced SERCA2 abundance resulted in decreased amplitude and decay rate of Ca(2+) transients, reduced SR Ca(2+) content, and decreased propensity for Ca(2+) wave development.

Alterations in trans-sarcolemmal and sarcoplasmic reticulum (SR) Ca(2+) fluxes may contribute to impaired cardiomyocyte contraction and relaxation in heart failure. We investigated the mechanisms underlying heart failure progression in mice with conditional, cardiomyocyte-specific excision of the SR Ca(2+)-ATPase (SERCA) gene. At 4 weeks following gene deletion (4-week KO) cardiac function remained near normal values. However, end-stage heart failure developed by 7 weeks (7-week KO) as systolic and diastolic performance declined. Contractions in isolated myocytes were reduced between 4- and 7-week KO, and relaxation was slowed. Ca(2+) transients were similarly altered. Reduction in Ca(2+) transient magnitude resulted from complete loss of SR Ca(2+) release between 4- and 7-week KO, due to loss of a small remaining pool of SERCA2. Declining SR Ca(2+) release was partly offset by increased L-type Ca(2+) current, which was facilitated by AP prolongation in 7-week KO. Ca(2+) entry via reverse-mode Na(+)-Ca(2+) exchange (NCX) was also enhanced. Up-regulation of NCX and plasma membrane Ca(2+)-ATPase increased Ca(2+) extrusion rates in 4-week KO. Diastolic dysfunction in 7-week KO resulted from further SERCA2 loss, but also impaired NCX-mediated Ca(2+) extrusion following Na(+) accumulation. Reduced Na(+)-K(+)-ATPase activity contributed to the Na(+) gain. Normalizing [Na(+)] by dialysis increased the Ca(2+) decline rate in 7-week KO beyond 4-week values. Thus, while SERCA2 loss promotes both systolic and diastolic dysfunction, Na(+) accumulation additionally impairs relaxation in this model. Our observations indicate that if cytosolic Na(+) gain is prevented, up-regulated Ca(2+) extrusion mechanisms can maintain near-normal diastolic function in the absence of SERCA2.

Sarco(endo)plasmic reticulum calcium ATPases (SERCA) are cellular pumps that transport Ca(2+) into the sarcoplasmic reticulum (SR). Serca2 is the most widely expressed gene family member. The very early embryonic lethality of Serca2(null) mouse embryos has precluded further evaluation of loss of Serca2 function in the context of organ physiology. We have generated mice carrying a conditional Serca2(flox) allele which allows disruption of the Serca2 gene in an organ-specific and/or inducible manner. The model was tested by mating Serca2(flox) mice with MLC-2v(wt/Cre) mice and with alphaMHC-Cre transgenic mice. In heterozygous Serca2(wt/flox)MLC-2v(wt/Cre) mice, the expression of SERCA2a and SERCA2b proteins were reduced in the heart and slow skeletal muscle, in accordance with the expression pattern of the MLC-2v gene. In Serca2(flox/flox) Tg(alphaMHC-Cre) embryos with early homozygous cardiac Serca2 disruption, normal embryonic development and yolk sac circulation was maintained up to at least embryonic stage E10.5. The Serca2(flox) mouse is the first murine conditional gene disruption model for the SERCA family of Ca(2+) ATPases, and should be a powerful tool for investigating specific physiological roles of SERCA2 function in a range of tissues and organs in vivo both in adult and embryonic stages.

The sarco(endo)plasmic reticulum calcium ATPase 2 (SERCA2) transports Ca(2+) from cytosol into the sarcoplasmic reticulum (SR) of cardiomyocytes, thereby maintaining the store of releasable Ca(2+) necessary for contraction. Reduced SERCA function has been linked to heart failure, and loss of SERCA2 in the adult mammalian heart would be expected to cause immediate severe myocardial contractile dysfunction and death. We investigated heart function in adult mice with an inducible cardiomyocyte-specific excision of the Atp2a2 (Serca2) gene (SERCA2 KO). Seven weeks after induction of Serca2 gene excision, the mice displayed a substantial reduction in diastolic function with a 5-fold increase in the time constant of isovolumetric pressure decay (tau). However, already at 4 weeks following gene excision less than 5% SERCA2 protein was found in myocardial tissue. Surprisingly, heart function was only moderately impaired at this time point. Tissue Doppler imaging showed slightly reduced peak systolic tissue velocity and a less than 2-fold increase in tau was observed. The SR Ca(2+) content was dramatically reduced in cardiomyocytes from 4-week SERCA2 KO mice, and Ca(2+) transients were predominantly generated by enhanced Ca(2+) flux through L-type Ca(2+) channels and the Na(+)-Ca(2+) exchanger. Moreover, equivalent increases in cytosolic [Ca(2+)] in control and SERCA2 KO myocytes induced greater cell shortening in SERCA2 KO, suggesting enhanced myofilament responsiveness. Our data demonstrate that SR-independent Ca(2+) transport mechanisms temporarily can prevent major cardiac dysfunction despite a major reduction of SERCA2 in cardiomyocytes.