Histidine-rich Ca-binding protein interacts with sarcoplasmic reticulum Ca-ATPase.

Depressed cardiac Ca cycling by the sarcoplasmic reticulum (SR) has been associated with attenuated contractility, which can progress to heart failure. The histidine-rich Ca-binding protein (HRC) is an SR component that binds to triadin and may affect Ca release through the ryanodine receptor. HRC overexpression in transgenic mouse hearts was associated with decreased rates of SR Ca uptake and delayed relaxation, which progressed to hypertrophy with aging. The present study shows that HRC may mediate part of its regulatory effects by binding directly to sarco(endo)plasmic reticulum Ca-ATPase type 2 (SERCA2) in cardiac muscle, which is confirmed by coimmunostaining observed under confocal microscopy. This interaction involves the histidine- and glutamic acid-rich domain of HRC (320-460 aa) and the part of the NH(2)-terminal cation transporter domain of SERCA2 (74-90 aa) that projects into the SR lumen. The SERCA2-binding domain is upstream from the triadin-binding region in human HRC (609-699 aa). Specific binding between HRC and SERCA was verified by coimmunoprecipitation and pull-down assays using human and mouse cardiac homogenates and by blot overlays using glutathione S-transferase and maltose-binding protein recombinant proteins. Importantly, increases in Ca concentration were associated with a significant reduction of HRC binding to SERCA2, whereas they had opposite effects on the HRC-triadin interaction in cardiac homogenates. Collectively, our data suggest that HRC may play a key role in the regulation of SR Ca cycling through its direct interactions with SERCA2 and triadin, mediating a fine cross talk between SR Ca uptake and release in the heart.

Overexpression of histidine-rich Ca-binding protein protects against ischemia/reperfusion-induced cardiac injury.

OBJECTIVE: The histidine-rich Ca-binding protein (HRC) is a Ca-storage protein in cardiac sarcoplasmic reticulum. Recent transgenic studies revealed that this protein inhibits the maximal rates of sarcoplasmic reticulum Ca-transport, leading to cardiac dysfunction. In view of the role of sarcoplasmic reticulum Ca-cycling in myocardial ischemia/reperfusion injury, we designed this study to gain further insight into the role of HRC during ischemia/reperfusion. METHODS AND RESULTS: The transgenic mouse model with cardiac-specific overexpression of HRC was utilized and cardiac contractile parameters were assessed before and after ischemia/reperfusion injury by Langendorff perfusion. After a 20-min stabilization period, the hearts were subjected to 40 min of global ischemia, followed by 60 min of reperfusion. We found that although transgenic (TG) hearts showed depressed cardiac function (25%) compared to wild types (WTs) at baseline, they exhibited better recovery of left ventricular developed pressure (86.6+/-2.6% in TGs vs. 58.3+/-4.0% in WTs of pre-ischemic values, P<0.05) and higher rates of contraction and relaxation after ischemia/reperfusion than WTs. This improvement was accompanied by smaller infarcts (23.1+/-1.7% in TGs vs. 41.1+/-2.5% in WTs of infarct region-to-risk region ratio, P<0.05) and lower creatine kinase release. Notably, the extent of apoptotic cell death was significantly attenuated, as evidenced by decreased DNA fragmentation, upregulation of the antiapoptotic protein Bcl-2, and downregulation of the active caspases (3, 9 and 12) following ischemia/reperfusion in TG hearts, compared with WTs. Extension of these studies to an in vivo model of 30-min myocardial ischemia, via coronary artery occlusion, followed by 24-h reperfusion, showed that the infarct region-to-risk region ratio was 9+/-0.9% in TGs, compared with 20.4+/-2.9% in WTs (P<0.05). CONCLUSIONS: Our findings suggest that increased cardiac HRC expression protects against ischemia/reperfusion injury in the heart, resulting in improved recovery of function and reduced infarction.

Histidine-rich Ca binding protein: a regulator of sarcoplasmic reticulum calcium sequestration and cardiac function.

Defects in the pathways that regulate cardiac sarcoplasmic reticulum (SR) calcium (Ca) cycling represent prime targets for driving the deterioration of function and progression to heart failure. We hypothesized that the histidine-rich Ca binding protein (HRC) in the SR may be involved in SR Ca cycling and that alterations in HRC levels would result in abnormal cardiac Ca homeostasis. In order to test this hypothesis, we generated transgenic mice with cardiac overexpression (3-fold) of HRC. Increased cardiac HRC levels were associated with impaired SR Ca uptake rates (35%) and attenuated cardiomyocyte Ca transient decay (38%), without alterations in peak Ca transients or SR Ca load. The depressed SR Ca sequestration was associated with attenuated rate of Ca extrusion via Na-Ca exchange. Triadin protein expression levels and L-type Ca channel current density were increased, while the channel inactivation kinetics were not altered. Impaired SR Ca uptake and delayed Ca decline rates triggered hypertrophy and compromised the heart's responses to increased stress by either hemodynamic overload or the aging process. By 18 months of age, cardiac remodeling deteriorated to congestive heart failure in transgenic mice. Collectively, these data suggest that HRC may be an integral regulatory protein involved in cardiac muscle SR Ca uptake and Ca homeostasis.

Sarcoplasmic reticulum Ca-ATPase-phospholamban interactions and dilated cardiomyopathy.

Dilated cardiomyopathy is a disease of the heart muscle resulting from a diverse array of conditions that damages the heart and impairs myocardial function. Heart failure occurs when the heart is unable to pump blood at a rate which can accommodate the heart muscle's metabolic requirements. Several signaling pathways have been shown to be involved in the induction of cardiac disease and heart failure. Many of these pathways are linked to cardiac sarcoplasmic reticulum (SR) Ca cycling directly or indirectly. A large body of evidence points to the central role of abnormal Ca handling by SR proteins, Ca-ATPase pump (SERCA2a) and phospholamban (PLN), in pathophysiological heart conditions, compromising the contractile state of the cardiomyocytes. This review summarizes studies which highlight the key role of these two SR proteins in the regulation of cardiac function, the significance of SERCA2a-PLN interactions using transgenic approaches, and the recent discoveries of human PLN mutations leading to disease states. Finally, we will discuss extrapolation of experimental paradigms generated in animal models to the human condition.

Regulation of myocardial function by histidine-rich, calcium-binding protein.

Impaired sarcoplasmic reticulum (SR) Ca release has been suggested to contribute to the depressed cardiac function in heart failure. The release of Ca from the SR may be regulated by the ryanodine receptor, triadin, junctin, calsequestrin, and a histidine-rich, Ca-binding protein (HRC). We observed that the levels of HRC were reduced in animal models and human heart failure. To gain insight into the physiological function of HRC, we infected adult rat cardiac myocytes with a recombinant adenovirus that contains the full-length mouse HRC cDNA. Overexpression (1.7-fold) of HRC in adult rat cardiomyocytes was associated with increased SR Ca load (28%) but decreased SR Ca-induced Ca release (37%), resulting in impaired Ca cycling and depressed fractional shortening (36%) as well as depressed rates of shortening (38%) and relengthening (33%). Furthermore, the depressed basal contractile and Ca kinetic parameters in the HRC-infected myocytes remained significantly depressed even after maximal isoproterenol stimulation. Interestingly, HRC overexpresssion was accompanied by increased protein levels of junctin (1.4-fold) and triadin (1.8-fold), whereas the protein levels of ryanodine receptor, calsequestrin, phospholamban, and sarco(endo)plasmic reticulum Ca-ATPase remained unaltered. Collectively, these data indicate that alterations in expression levels of HRC are associated with impaired cardiac SR Ca homeostasis and contractile function.

Increased particulate partitioning of PKC epsilon reverses susceptibility of phospholamban knockout hearts to ischemic injury.

Cytosolic Ca(2+) overload is a critical mediator of myocardial damage following cardiac ischemia-reperfusion. It has therefore been proposed that normalization of sarcoplasmic reticulum Ca(2+) cycling through inhibition or ablation of the Ca(2+) ATP-ase inhibitor phospholamban (PLN), which shows promise as a treatment for heart failure, could be beneficial in ischemic heart disease. However, a recent study has shown that globally ischemic PLN-deficient hearts exhibit increased ischemic injury, with impaired contractile, ATP, and phosphocreatine recoveries, compared to wild-type hearts. Since protein kinase C (PKC) family members are widely recognized as mediators of both post-ischemic injury and ischemic preconditioning, we assessed PKC levels in PLN-deficient hearts. Compared to genetically normal hearts, PLN-deficient hearts exhibited diminished particulate partitioning of PKC, a known cardioprotective PKC isoform, without alterations in the levels of membrane-associated PKC delta nor PKC alpha. To determine if decreased particulate partitioning of cardioprotective PKC epsilon was a cause of increased ischemic injury in PLN-deficient hearts, PLN-deficient mice were mated with mice expressing a myocardial-specific PKC epsilon translocation activator peptide, pseudo-epsilon receptor for activated kinase C (psi epsilon RACK). In psi epsilon RACK/PLN knockout (KO) hearts, PKC epsilon translocation to membranous cellular structures was augmented and this was associated with a significant acceleration of post-ischemic contraction and relaxation rates, as well as reduction of creatine phosphokinase release, compared to PLN-deficient hearts. Importantly, post-ischemic functional recovery reached pre-ischemic hyperdynamic values in psi epsilon RACK/PLN KO hearts, indicating super-rescue by the combination of PLN ablation and psi epsilon RACK expression. These findings suggest that diminished PKC epsilon particulate partitioning in PLN-deficient hearts is associated with attenuated contractile recovery upon ischemia-reperfusion and that increased translocation of PKC to membranous cellular structures confers full cardioprotection.

Hierarchy of polymorphic variation and desensitization permutations relative to beta 1- and beta 2-adrenergic receptor signaling.

Agonist-promoted desensitization of G-protein-coupled receptors results in partial uncoupling of receptor from cognate G-protein, a process that provides for rapid adaptation to the signaling environment. This property plays important roles in physiologic and pathologic processes as well as therapeutic efficacy. However, coupling is also influenced by polymorphic variation, but the relative impact of these two mechanisms on signal transduction is not known. To determine this we utilized recombinant cells expressing the human beta(1)-adrenergic receptor (beta(1)AR) or a gain-of-function polymorphic variant (beta(1)AR-Arg(389)), and the beta(2)-adrenergic receptor (beta(2)AR) or a loss-of-function polymorphic receptor (beta(2)AR-Ile(164)). Adenylyl cyclase activities were determined with multiple permutations of the possible states of the receptor: genotype, basal, or agonist stimulated and with or without agonist pre-exposure. For the beta(1)AR, the enhanced function of the Arg(389) receptor underwent less agonist-promoted desensitization compared with its allelic counterpart. Indeed, the effect of polymorphic variation on absolute adenylyl cyclase activities was such that desensitized beta(1)AR-Arg(389) signaling was equivalent to non-desensitized wild-type beta(1)AR; that is, the genetic component had as much impact as desensitization on receptor coupling. In contrast, the enhanced signaling of wild-type beta(2)AR underwent less desensitization compared with beta(2)AR-Ile(164), thus the heterogeneity in absolute signaling was markedly broadened by this polymorphism. Inverse agonist function was not affected by polymorphisms of either subtype. A general model is proposed whereby up to 10 levels of signaling by G-protein-coupled receptors can be present based on the influences of desensitization and genetic variation on coupling.