A mouse model carrying a null mutation in one copy of the sarcoplasmic reticulum (SR) Ca 2؉ -ATPase isoform 2 (SERCA2) gene, in which SERCA2 protein levels are reduced by ϳ35%, was used to investigate the effects of decreased SERCA2 level on intracellular Ca 2؉ homeostasis and contractile properties in isolated cardiomyocytes. When compared with wild-type controls, SR Ca 2؉ stores and Ca 2؉ release in myocytes of SERCA2 heterozygous mice were decreased by ϳ40 -60% and ϳ30 -40%, respectively, and the rate of myocyte shortening and relengthening were each decreased by ϳ40%. However, the rate of Ca 2؉ transient decline () was not altered significantly, suggesting that compensation was occurring in the removal of Ca 2؉ from the cytosol. Phospholamban, which inhibits SERCA2, was decreased by ϳ40% in heterozygous hearts, and basal phosphorylation of Ser-16 and Thr-17, which relieves the inhibition, was increased ϳ2-and 2.1-fold. These results indicate that reduced expression and increased phosphorylation of phospholamban provides compensation for decreased SERCA2 protein levels in heterozygous heart. Furthermore, both expression and current density of the sarcolemmal Na ؉ -Ca 2؉ exchanger were up-regulated. These results demonstrate that a decrease in SERCA2 levels can directly modify intracellular Ca 2؉ homeostasis and myocyte contractility. However, the resulting deficit is partially compensated by alterations in phospholamban/SERCA2 interactions and by up-regulation of the Na ؉ -Ca 2؉ exchanger.In heart, muscle relaxation is largely dependent on the action of the sarcoplasmic reticulum (SR) 1 Ca 2ϩ ATPase (SERCA)to resequester cytosolic calcium released during contraction. Increased activity of SERCA, either by transgenic overexpression of SERCA isoforms in the heart (1-3) or by ablation of its regulatory protein, phospholamban (PLB) (4), has been shown to enhance cardiac rates of contraction and relaxation (1-4). To examine the effects of decreased SERCA2 activity on cardiac function, we have recently developed a transgenic mouse model with a null allele of the SERCA2 gene (5). Although complete loss of SERCA function in homozygous animals is embryonic lethal, disruption of one copy of the SERCA2 gene results in decreased cardiac SERCA2 mRNA (ϳ45%), protein (ϳ35%), and SR Ca 2ϩ uptake (ϳ35%) (5). These changes are associated in vivo with impaired cardiac performance (5). Because SERCA2 activity controls both the rate of calcium removal and the amount of calcium stores available within the SR, we hypothesize that the level of SERCA2 activity is a critical determinant of cardiac contractility. Therefore, one goal of this study is to determine if reduced SERCA2 levels compromise cardiac contractility by directly altering calcium handling and contractile functions of individual myocytes during excitationcontraction coupling.During excitation-contraction coupling, Ca 2ϩ entry through the L-type Ca 2ϩ channel activates Ca 2ϩ release from SR Ca 2ϩ stores, via the ryanodine receptor (RyR). This rise in cytosolic Ca 2ϩ i...

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.The sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA) 1 family of proteins is encoded by three separate genes: SERCA1, SERCA2, and SERCA3. Each of these genes is transcribed in a tissue-specific manner, and alternate splicing results in at least six different isoforms. SERCA1a is expressed in adult fast-twitch skeletal muscle (1, 2). SERCA2a is expressed at high levels in cardiac and slow-twitch skeletal muscle (3). SERCA2b, the so-called "housekeeping" isoform, is expressed virtually in every cell type (4, 5). The SERCA3 isoforms are expressed in specialized non-muscle cells (6, 7). SERCA2b is ubiquitously expressed; it is responsible for maintaining intracellular Ca 2ϩ stores and plays an important role in regulating Ca 2ϩ signaling in vital tissues including neurons, pancreas, and other secretory cells. Although SERCA2b protein is detected in the heart tissue, it has not been shown to be expressed specifically in cardiac myocytes, and the role of SERCA2b in muscle has never been clearly defined. For example, in pancreatic and salivary gland cells, specific and polarized expression of SERCA isoforms has been observed (8). In pancreatic acinar cells, SERCA2a is found in the luminal pole, whereas SERCA2b is expressed in the basal pole and nuclear envelope. In salivary gland cells, SERCA2b is found in the luminal pole, whereas SERCA3 is expressed in the basal pole. By analogy to its role in other cell types, SERCA2b might be expected to play such a unique role in cardiac myocytes. No such intracellular compartmentalization or ...

Phospholamban (PLB), a protein localized in the sarcoplasmic reticulum (SR), inhibits the SR Ca2+-ATPase; phosphorylation of PLB relieves this inhibition. We previously reported significant differences in contractility in aorta from mice in which the gene for PLB was ablated (PLB−). In this study, we measured intracellular Ca2+concentration ([Ca2+]i) with fura 2 in the intact mouse aorta to more directly test the hypothesis that these changes are ascribable to altered SR function in vivo. Ten micromoles per liter of the α-agonist phenylephrine (PE) increased [Ca2+]imonotonically to a steady state in the wild-type aorta. In contrast, in PLB− aorta there was an initial rapid increase to a peak [Ca2+]i, which then decreased to a steady state that was lower than that in the wild type. Upon removal of the stimulus (either PE or KCl), the decrease in [Ca2+]iwas two times as fast in the PLB− as in the wild-type aorta. There were no significant differences between PLB− and wild-type aortas in the concentration vs. force relations or the time courses of relaxation in response to forskolin or sodium nitroprusside. Interestingly, stimulation of the cAMP pathway before cGMP pathway activation resulted in a significant increase in sensitivity and a difference in relaxation parameters between PLB− and wild-type aortas. Western blot analysis indicated that the PLB-to-sarcoendoplasmic reticulum Ca2+ATPase ratio in the mouse aorta was similar to that in the heart; 20-fold more aortic than heart homogenate was required to achieve a similar level of immunoreactivity. Our data indicate that PLB can play a major role in modulating smooth muscle [Ca2+]ibut only a minor role, if any, in cyclic nucleotide-mediated relaxation.

Changes in calcium (Ca2+) regulation contribute to loss of contractile function in dilated cardiomyopathy. Clinical treatment using β-adrenergic receptor antagonists (β-blockers) slows deterioration of cardiac function in end-stage heart failure patients; however, the effects of β-blocker treatment on Ca2+ dynamics in the failing heart are unknown. To address this issue, tropomodulin-overexpressing transgenic (TOT) mice, which suffer from dilated cardiomyopathy, were treated with a nonselective β-receptor blocker (5 mg · kg-1 · day-1 propranolol) for 2 wk. Ca2+ dynamics in isolated cardiomyocytes of TOT mice significantly improved after treatment compared with untreated TOT mice. Frequency-dependent diastolic and Ca2+ transient amplitudes were returned to normal in propranolol-treated TOT mice and but not in untreated TOT mice. Ca2+ kinetic measurements of time to peak and time decay of the caffeine-induced Ca2+ transient to 50% relaxation were also normalized. Immunoblot analysis of untreated TOT heart samples showed a 3.6-fold reduction of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), whereas Na+/Ca2+ exchanger (NCX) concentrations were increased 2.6-fold relative to nontransgenic samples. Propranolol treatment of TOT mice reversed the alterations in SERCA and NCX protein levels but not potassium channels. Although restoration of Ca2+ dynamics occurred within 2 wk of β-blockade treatment, evidence of functional improvement in cardiac contractility assessed by echocardiography took 10 wk to materialize. These results demonstrate that β-adrenergic blockade restores Ca2+ dynamics and normalizes expression of Ca2+-handling proteins, eventually leading to improved hemodynamic function in cardiomyopathic hearts.

Cardiac ATP-sensitive K+ (KATP) channels (SUR2A plus Kir6.2) couple the metabolic state of the myocyte to its electrical activity via a mechanism that is not well understood. Recent pharmacological evidence suggests that KATP channels may mediate ischemic preconditioning. However, there is no potent pharmaceutical agent that specifically blocks the sarcolemmal KATP channel without significant effects on other cellular proteins. As a molecular tool, the GFG sequence in the H5 loop of the murine Kir6.2 channel was mutated to AFA. This mutated channel subunit (6.2AFA) suppressed wild-type Kir6.2 (6.2WT) channel current in a dominant-negative manner: when co-expressed with SUR2A and 6.2WT, whole-cell KATP current recorded from HEK cells was greatly attenuated. The 6.2AFA subunit also co-assembled with endogenous subunits in both smooth-muscle-derived A10 cells and rat neonatal ventricular myocytes, resulting in a significant reduction of current compared with that recorded from non-transfected or mock-transfected cells (<15% of control for both cell types). This study shows that mutation of GFG-->AFA in the putative pore-forming region of Kir6.2 acts in a dominant-negative manner to suppress current in heterologous systems and in native cells.