THE Ca²⁺ ATPase OF CARDIAC SARCOPLASMIC RETICULUM: PHYSIOLOGICAL ROLE AND RELEVANCE TO DISEASES

Abstract

The Ca²⁺ ATPase of sarcoplasmic reticulum has a prominent role in excitation/contraction coupling of cardiac muscle, as it induces relaxation by sequestering Ca²⁺ from the cytoplasm. The stored Ca²⁺ is in turn released to trigger contraction. We review here experiments demonstrating that in cardiac myocytes Ca²⁺ signaling and contractile activation are strongly altered by pharmacological inhibiton or transcriptional downregulation of SERCA. On the other hand, kinetics and intensity of Ca²⁺ signaling are improved by SERCA overexpression following delivery of exogenous cDNA by adenovirus vectors. Experiments on adrenergic hypertrophy demonstrate SERCA downregulation, consistent with its pathogenetic involvement in cardiac hypertrophy and failure, as also shown in other experimental models and clinical studies. Compensation by alternate Ca²⁺ signaling proteins, including functional activation and increased expression of Na⁺/Ca²⁺ exchanger and TrpC proteins has been observed. These compensatory mechanisms, including calcineurin activation, remain to be clarified and are a most important subject of current studies.

On occasion of Professor Ebashi’s visit to the Cardiovascular Research Institute of the University of California in San Francisco, the earliest preparations of vesicular fragments of cardiac sarcoplasmic reticulum (SR) were obtained under his guidance (1). A warm memory remains with us of Professor Ebashi’s influence and guidance through many hours in the laboratory, continuing at the end of day with stimulating and inspiring discussions at dinner in various San Francisco restaurants. At that time, SR membrane vesicles were referred to as “Relaxing Factor” since they prevented “superprecipitation” (i.e., contraction analog) of native actomyosin (containing myosin, actin and the troponin complex) upon addition of ATP. It was soon established that this effect was produced by ATP dependent sequestration of Ca²⁺ by the vesicles from the reaction medium. In fact, the same relaxing effect could be produced simply by Ca²⁺ chelation with EGTA added to the medium. The functional behavior of isolated cardiac SR vesicle in vitro turned out to be similar to that exhibited by SR vesicles isolated from skeletal muscle (2, 3), due to the Ca²⁺ activated Sarco-Endoplasmic Reticulum ATPase (SERCA) which is a prominent protein component of both preparations. The catalytic and transport cycle of SERCA includes ATP utilization by formation of a phosphorylated enzyme intermediate, translocation of bound Ca²⁺ across the SR membrane against a concentration gradient, and final hydrolytic cleavage of Pi from the phosphoenzyme intermediate (4). We review here information presently available on the role of SERCA in physiology and diseases of heart muscle, discovered by several laboratories working in the field, and illustrated by experimental results obtained in our own laboratory.

SERCA and Ca²⁺ signaling in cardiac muscle

The cardiac SR ATPase (SERCA2a) is encoded by the (human nomenclature) ATP2A2 gene, which also yields the SERCA2b isoform found in the endoplasmic reticulum of most cells (5, 6). It is by now well established that SERCA2 plays an important role in relaxation of heart muscle, as it sequesters cytosolic Ca²⁺ by active transport into intracellular stores delimited by the SR membranes. The stored Ca²⁺ is in turn released into the cytoplasm upon membrane excitation to induce contraction. This can be convincingly demonstrated on primary cultures of cardiac myocytes, as they provide a rather simple system to study complementary features of SERCA transport activity and cytosolic Ca²⁺ signaling.

The cytosolic Ca²⁺ signaling transients observed in myocytes subjected to electric stimuli include a rapid rise followed by a slower decay (Fig 1). The rapid Ca²⁺ rise does not show significant dependence on temperature, as expected of passive flux through a channel. On the contrary, the rate of decay exhibits strong temperature dependence, similar to that exhibited by the transport activity of isolated SR vesicles (Fig 1). This parallel behavior is consistent with dependence of the decay phase of Ca²⁺ transients on active transport by the SERCA enzyme (7).

Fig 1. Temperature dependence of cytosolic Ca²⁺ transients and active Ca²⁺ transport by cardiac SR vesicles.

The Ca²⁺ transients were recorded using a fluorescence indicator in cultured neonatal rat myocytes (A). ATP dependent Ca²⁺ active transport (B) was measured with homogenized myocytes using radioactive tracer (7).

The dependence of cytosolic Ca²⁺ signaling on transport by SERCA can be demonstrated directly by inhibiting its catalytic and transport activity (8) with thapsigargin (TG), a plant derived sesquiterpene lactone. Specific inhibition of SERCA is obtained in situ by exposing cardiac myocytes to nanomolar concencentrations of TG in the culture medium (Fig 2). Such low concentrations of TG inhibit specifically SERCA activity, without apparent effects on cell growth or other cell characteristics (9). An alternative experimental approach is downregulation of SERCA expression by the use of short interfering RNA (10), with consequent reduction of overall transport activity (Fig 2). Additional evidence is obtained in myocytes subjected to adrenergic hypertrophy (9), as significant reduction of SERCA expression is produced relative to general protein expression. In all cases, reduction of Ca²⁺ transport activity, due to either SERCA inhibition or down regulation, results in pronounced alterations of cytosolic transients and marked slowing of the decay phase (Fig 3). It is of interest that SERCA inhibition with TG in myocytes, is followed by compensatory increase in Na⁺/Ca²⁺ exchange activity (Fig 4), as a compensatory mechanism to eliminate cytosolic Ca²⁺ through the plasma membrane. In fact, if Na⁺/Ca²⁺ exchange is prevented by placing the myocytes in Na⁺ free medium, the basal (“diastolic”) level of cytosolic Ca²⁺, between consecutive stimuli, is progressively increased (Fig 4).

Fig 2. Protein levels and ATP dependent Ca²⁺ transport in homogenates of myocytes exposed to 20 µM PE, siSERCA2 or 10 nM TG.

Control myocytes (1) are compared to myocytes exposed to PE for three (2) or seven days (3), or siSERCA2 for three days (4), or 10 nM TG (5) for three days. Protein levels were measured by Western blotting and compared to the levels of GAPDH protein. ATP dependent calcium transport was measured using homogenates of cultured cardiac myocytes, harvested after 3 or 7 days of treatment as specified above (9).

Fig 3. Alteration of Ca²⁺ signaling kinetics in myocytes exposed to PE, SERCA2 short interfering RNA or TG.

The myocytes were exposed to 20 µM PE for seven days, or to siSERCA2 for three days, or to 10 nM TG for three days. Cytosolic Ca²⁺ transients were measured in single cells using fluo-4. The cells were subjected to field stimulation (1 Hz pulsing). Each trace represents the average of transients obtained from 30 to 70 cells over 5 different preparations. The fluorescence signal was normalized to the maximal change per each transient (9).

Fig 4. Compensatory increase in Na⁺ /Ca²⁺ exchange activity following SERCA inhibition by TG in cardiac myocytes.

Control myocytes (upper panel) and myocytes exposed to 10 nM TG for three days (lower Panel) were subjected to a series of voltage clamp depolarizations to −10 mv at 2 sec intervals. Ca²⁺ transients were then measured in Na⁺ free bathing solution, thereby eliminating Na⁺ /Ca²⁺ exchange. It is clear that, in the absence of Na⁺/Ca²⁺ exchange, the myocytes with inhibited SERCA show a progressive increase of the basal cytosolic Ca²⁺ level as a consequence of consecutive stimuli (9).

As a further demonstration of SERCA involvements in Ca²⁺ transient kinetics, faster decay of the transients can be obtained by SERCA overexpression, encoded by exogenous cDNA. For this purpose, we have used adenoviral vectors for efficient introduction of exogenous SERCA cDNA in cardiac myocytes, under control of constitutive or cardiac cell specific promoters (11). Most importantly, it can be shown by the use of diverse SERCA isoforms and specific antibodies (12), that the protein encoded by the exogenous cDNA is targeted exactly to the same membrane location as the protein encoded by the endogenous gene (Fig 5). In all cases, the resulting effect on Ca²⁺ transients is a shorter decay phase, as expected from a faster Ca²⁺ removal from the cytoplasm (Fig 6).

Fig 5. Intracellular targeting of SERCA1 gene product following delivery of exogenous cDNA by means of adenovirus vector under control of cardiac cell specific promoter.

Colocalization of exogenous SERCA1 with endogenous SERCA2 is demonstrated by specific immunostaining for endogenous SERCA2 (A and D), exogenous SERCA 1 (B and E), and combined staining forn SERCA2 and SERCA1 (C and F). Scale bars correspond to 10 µm (12).

Fig 6. Ca²⁺ signaling transients in control cardiac myocytes, and following overexpression of exogenous SERCA.

Cytosolic Ca²⁺ transients were measured in chicken embryo or neonatal rat myocytes using fluo-4. Myocytes overexpressing exogenous SERCA1 or SERCA2 (following infection with adenovirus vectors) are compared with control myocytes. The cells were subjected to field stimulation (1 Hz pulsing). Each trace represents the average of transients obtained from 15–30 cells over 3–5 different preparations. The fluorescence signal was normalized to the maximal change per each transient (12).

Physiologic and pathogenetic role of SERCA in excitation-contraction coupling

An intriguing question is whether, in addition to kinetic effects, the maximal level of Ca²⁺ stored within the lumen of the SR is affected by changes in overall SERCA activity. Assuming the same internal volume delimited by the SR membrane, the maximal Ca²⁺ concentration that can be possibly reached in the lumen of SR depends on Ca²⁺ dissociation constant from the ATPase inward oriented Ca²⁺ binding sites following utilization of ATP. In principle, this limit is independent of the overall Ca²⁺ transport velocity. On the other hand, if the frequency of consecutive Ca²⁺ release stimuli is increased, the time interval between stimuli becomes a limiting factor, and the overall transport velocity then determines the level of maximal filling. This is in fact observed when myocytes are subjected to trains of stimuli with progressively shorter intervals, whereby the amount of released Ca²⁺ becomes progressively smaller, as it is limited by the luminal Ca²⁺ load reached within the short interval. In this case, compensation is obtained by overexpression of exogenous SERCA, as a greater number of SERCA molecules allows a higher overall transport velocity and a more effective filling of the SR lumen within a limited time interval between stimuli (Fig 7).

Fig 7. Ca²⁺ signaling transients (A) following stimuli at progressively shorter.

intervals in control myocytes and following overexpression of exogenous SERCA. It is shown that the amount of released Ca²⁺ is reduced (B) as the interval between consecutive stimuli is shorter, indicating a time limit for Ca²⁺ refilling of SR stores between stimuli. This limit is significantly overcome by overexpression of exogenous SERCA1 (7)

With regard to the relevance of Ca²⁺ transport to contractile activity, it has been clearly shown that specific inhibition of SERCA by TG in situ, interferes (13) with the contractile response of cardiac myocytes to membrane excitation. This effect is certainly relevant to cardiac pathology. In fact, many experimental and clinical studies indicate that inadequate function of the Ca²⁺ transport ATPase (SERCA2) and the consequent alterations of cytosolic Ca²⁺ signaling are pathogenetic features of cardiac hypertrophy and failure (14 – 24). Even expression of SERCA isoforms with higher than normal Ca²⁺ affinity can lead to cardiac hypertrophy and failure (25).

An important feature of SERCA2 is its regulation by phospholamban in cardiac muscle, as originally described by Kirchberger et al. (26) and subsequently studied extensively in several laboratories, especially by E.G. Kranias (27). Phospholamban is a small protein that interacts with the transmembrane and stalk regions of the SERCA protein. The functional effect resulting from this interaction is a displacement of the ATPase activation curve to a higher Ca²⁺ concentrations range, therefore yielding lower activity at relevant cytosolic Ca²⁺ concentrations (28). This effect is overcome by phospholamban phosphorylation following adrenergic activation of protein kinase, thereby providing an elegant explanation for the increased cardiac contraction upon sympathetic discharge. Numerous and elegant studies with transgenic animals have shown convincingly that phospholamban is a key determinant of cardiac function and dysfunction (29). In addition, specific mutations in the human phospholamban gene may result in severe cardiomyopathies (30).

The evidence indicating that deficiencies of SERCA2 expression, function and regulation may be a significant factor in the pathogenesis of cardiac hypertrophy and failure ( 14 – 24), has led to attempts to relieve related shortcomings by over-expression through introduction of exogenous SERCA cDNA (31 – 37). On the other hand, it is now becoming apparent that regulation of endogenous expression is an important factor to be considered. In the experimental setting, it is clear that myocytes undergoing adrenergic hypertrophy (38) under-express SERCA, as the enzyme does not seem to be included in the hypertrophy transcriptional program (9). In addition, alterations of cytosolic Ca²⁺ homeostasis trigger over-expression of other Ca²⁺ handing proteins such as Na⁺/Ca²⁺ exchanger and the TRPC proteins (10). Calcineurin, a Ca²⁺ /calmodulin activated phosphatase and transciptiona activator (39, 40) that plays an important role in heart development and remodeling (41, 42), is likely to be involved in adaptive responses to alterations of cytosolic Ca²⁺ homeostasis (43 – 45). However, the consequences of its activation are likely to be interwoven with additional mechanisms of transcriptional regulation (46) that may enhance or counteract the overall effect, directing it to expression of specific proteins, and excluding others. Presently, clarification of adaptive mechanisms for up- and down-regulation of endogenous SERCA and other Ca²⁺ handling proteins is a most promising endeavor in order to gain understanding and hopefully improving treatment of cardiac hypertrophy and failure.