The protein kinase C (PKC) family of serine/threonine kinases functions downstream of nearly all membrane-associated signal transduction pathways. Here we identify PKC-alpha as a fundamental regulator of cardiac contractility and Ca(2+) handling in myocytes. Hearts of Prkca-deficient mice are hypercontractile, whereas those of transgenic mice overexpressing Prkca are hypocontractile. Adenoviral gene transfer of dominant-negative or wild-type PKC-alpha into cardiac myocytes enhances or reduces contractility, respectively. Mechanistically, modulation of PKC-alpha activity affects dephosphorylation of the sarcoplasmic reticulum Ca(2+) ATPase-2 (SERCA-2) pump inhibitory protein phospholamban (PLB), and alters sarcoplasmic reticulum Ca(2+) loading and the Ca(2+) transient. PKC-alpha directly phosphorylates protein phosphatase inhibitor-1 (I-1), altering the activity of protein phosphatase-1 (PP-1), which may account for the effects of PKC-alpha on PLB phosphorylation. Hypercontractility caused by Prkca deletion protects against heart failure induced by pressure overload, and against dilated cardiomyopathy induced by deleting the gene encoding muscle LIM protein (Csrp3). Deletion of Prkca also rescues cardiomyopathy associated with overexpression of PP-1. Thus, PKC-alpha functions as a nodal integrator of cardiac contractility by sensing intracellular Ca(2+) and signal transduction events, which can profoundly affect propensity toward heart failure.

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.

No abstract

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.

No abstract

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 ␤ 1 -adrenergic receptor (␤ 1 AR) or a gain-of-function polymorphic variant (␤ 1 AR-Arg 389 ), and the ␤ 2 -adrenergic receptor (␤ 2 AR) or a loss-of-function polymorphic receptor (␤ 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 ␤ 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 ␤ 1 AR-Arg 389 signaling was equivalent to non-desensitized wild-type ␤ 1 AR; that is, the genetic component had as much impact as desensitization on receptor coupling. In contrast, the enhanced signaling of wild-type ␤ 2 AR underwent less desensitization compared with ␤ 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.Like a number of other G-protein-coupled receptors, the ␤ 1 -and ␤ 2 -adrenergic receptors (␤ 1 AR and ␤ 2 AR, 1 respectively) undergo desensitization during continuous exposure to agonist.Such desensitization occurs maximally after several minutes of agonist exposure and is due to decreased interaction with G s , which is evoked by receptor phosphorylation (1). Thus the signal transduction of these receptors can be characterized as one of two potential conditions or states, defined here as "control" (no recent exposure to agonist) and "desensitized." However, we have recently shown that an alteration in receptor-G s coupling can also be imposed by genetic mechanisms. A single nucleotide polymorphism found in the ␤ 1 AR gene in the human population (2, 3) results in either Gly or Arg being encoded at amino acid position 389 of the proximal portion of the cytoplasmic tail. In studies using transfected cells with equivalent expression of the two receptors, the ␤ 1 AR-Arg 389 displays an increase in G s coupling compared with ␤ 1 AR-Gly 389 (2). So, one can consider that the human ␤ 1 AR can exist in four agoniststimulated states: Gly 389 control and desensitized, and Arg 389 control and desensitized. And, since basal (non-ag...

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.

No abstract