Compensatory Mechanisms Associated With the Hyperdynamic Function of Phospholamban-Deficient Mouse Hearts

Abstract

Phospholamban ablation is associated with significant increases in the sarcoplasmic reticulum Ca(2+)-ATPase activity and the basal cardiac contractile parameters. To determine whether the observed phenotype is due to loss of phospholamban alone or to accompanying compensatory mechanisms, hearts from phospholamban-deficient and age-matched wild-type mice were characterized in parallel. There were no morphological alterations detected at the light microscope level. Assessment of the protein levels of the cardiac sarcoplasmic reticulum Ca(2+)-ATPase, calsequestrin, myosin, actin, troponin I, and troponin T revealed no significant differences between phospholamban-deficient and wild-type hearts. However, the ryanodine receptor protein levels were significantly decreased (25%) upon ablation of phospholamban, probably in an attempt to regulate the release of Ca2+ from the sarcoplasmic reticulum, which had a significantly higher diastolic Ca2+ content in phospholamban-deficient compared with wild-type hearts (16.0 +/- 2.2 versus 8.6 +/- 1.0 mmol Ca2+/kg dry wt, respectively). The increases in Ca2+ content were specific to junctional sarcoplasmic reticulum stores, as there were no alterations in the Ca2+ content of the mitochondria or A band. Assessment of ATP levels revealed no alterations, although oxygen consumption increased (1.6-fold) to meet the increased ATP utilization in the hyperdynamic phospholamban-deficient hearts. The increases in oxygen consumption were associated with increases (2.2-fold) in the active fraction of the mitochondrial pyruvate dehydrogenase, suggesting increased tricarboxylic acid cycle turnover and ATP synthesis. 31P nuclear magnetic resonance studies demonstrated decreases in phosphocreatine levels and increases in ADP and AMP levels in phospholamban-deficient compared with wild-type hearts. However, the creatine kinase activity and the creatine kinase reaction velocity were not different between phospholamban-deficient and wild-type hearts. These findings indicate that ablation of phospholamban is associated with downregulation of the ryanodine receptor to compensate for the increased Ca2+ content in the sarcoplasmic reticulum store and metabolic adaptations to establish a new energetic steady state to meet the increased ATP demand in the hyperdynamic phospholamban-deficient hearts.

Rescue of Contractile Parameters and Myocyte Hypertrophy in Calsequestrin Overexpressing Myocardium by Phospholamban Ablation

“…The protein levels of the SR Ca 2ϩ -ATPase were increased in parallel with up-regulation of phospholamban in CSQ OE hearts, but ablation of phospholamban prevented the compensatory up-regulation of the SR Ca 2ϩ -ATPase (Table I). The ryanodine receptor levels, which were decreased in CSQ OE hearts, were further reduced in CSQ OE / PLB KO hearts, similar to previous observations in phospholamban-deficient mice (16). Furthermore, the levels of GRP78 (78-kDa glucose-regulated protein, also known as BiP) and calreticulin, which are Ca 2ϩ -binding proteins localized in the lumen of the endoplasmic reticulum (ER) or the SR, were up-regulated by 3.4-and 2.0-fold in CSQ OE , respectively.…”

“…Decreases in the levels of phospholamban or increases in its phosphorylation status result in an increase in the apparent Ca 2ϩ affinity of the SR Ca 2ϩ -ATPase, and augment cardiac contractile parameters. The mechanisms underlying these regulatory effects of phospholamban have been suggested to reflect facilitation of SR Ca 2ϩ transport, Ca 2ϩ loading, and Ca 2ϩ release (13)(14)(15)(16)(17)(18)(19). These studies also indicate that the SR Ca 2ϩ load is one of the major determinants of myocardial contractility (18,19).…”

Superinhibition of Sarcoplasmic Reticulum Function by Phospholamban Induces Cardiac Contractile Failure

“…Biochemical Assays-Quantitative immunoblotting of cardiac homogenates was used to determine the levels of PLB and SR calciumhandling proteins (25). Cardiac gene expression was assayed by dot blot analysis of total RNA (10 g) using 32 P-labeled oligonucleotide probes (25).…”

“…Cardiac gene expression was assayed by dot blot analysis of total RNA (10 g) using 32 P-labeled oligonucleotide probes (25). Oxalate-supported Ca 2ϩ uptake in cardiac homogenates was measured by a modified Millipore filtration technique (15).…”

Biophysical interaction between phospholamban and protein phosphatase 1 regulatory subunit GM

“…The physiological role of PLB on the activity of the calcium ATPase has been demonstrated using transgenic models in which the intracellular calcium transient and the myocardial relaxation are directly related to the level of expressed PLB [1]. Similarly, agents that inhibit the interaction between PLB and SERCA 2a, such as ellagic acid, can increase the activity of the calcium pump [2].…”