Differential integration of Ca2+-calmodulin signal in intact ventricular myocytes at low and high affinity Ca2+-calmodulin targets.
ABSTRACT: Cardiac myocyte intracellular calcium varies beat-to-beat and calmodulin (CaM) transduces Ca2+ signals to regulate many cellular processes (e.g. via CaM targets such as CaM-dependent kinase and calcineurin). However, little is known about the dynamics of how CaM targets process the Ca2+ signals to generate appropriate biological responses in the heart. We hypothesized that the different affinities of CaM targets for the Ca2+-bound CaM (Ca2+-CaM) shape their actions through dynamic and tonic interactions in response to the repetitive Ca2+ signals in myocytes. To test our hypothesis, we used two fluorescence resonance energy transfer-based biosensors, BsCaM-45 (Kd = approximately 45 nm) and BsCaM-2 (Kd = approximately 2 nm), to monitor the real time Ca2+-CaM dynamics at low and high affinity CaM targets in paced adult ventricular myocytes. Compared with BsCaM-2, BsCaM-45 tracks the beat-to-beat Ca2+-CaM alterations more closely following the Ca2+ oscillations at each myocyte contraction. When pacing frequency is raised from 0.1 to 1.0 Hz, the higher affinity BsCaM-2 demonstrates significant elevation of diastolic Ca2+-CaM binding compared with the lower affinity BsCaM-45. Biochemically detailed computational models of Ca2+-CaM biosensors in beating cardiac myocytes revealed that the different Ca2+-CaM binding affinities of BsCaM-2 and BsCaM-45 are sufficient to predict their differing kinetics and diastolic integration. Thus, data from both experiments and computational modeling suggest that CaM targets with low versus high Ca2+-CaM affinities (like CaM-dependent kinase versus calcineurin) respond differentially to the same Ca2+ signal (phasic versus integrating), presumably tuned appropriately for their respective and distinct Ca2+ signaling pathways.
Project description:Calmodulin (CaM) mediates Ca-dependent regulation of numerous pathways in the heart, including CaM-dependent kinase (CaMKII) and calcineurin (CaN), yet the local Ca(2+) signals responsible for their selective activation are unclear. To assess when and where CaM, CaMKII, and CaN may be activated in the cardiac myocyte, we integrated new mechanistic computational models of CaM, CaMKII, and CaN with the Shannon-Bers model of excitation-contraction coupling in the rabbit ventricular myocyte. These models are validated with independent in vitro data. In the intact myocyte, model simulations predict that CaM is highly activated in the dyadic cleft during each beat, but not appreciably in the cytosol. CaMKII-delta(C) was almost insensitive to cytosolic Ca due to relatively low CaM affinity. Dyadic cleft CaMKII exhibits dynamic frequency-dependent responses to Ca, yet autophosphorylates only when local phosphatases are suppressed. In contrast, dyadic cleft CaN in beating myocytes is predicted to be constitutively active, whereas the extremely high affinity of CaN for CaM allows gradual integration of small cytosolic CaM signals. Reversing CaM affinities for CaMKII and CaN also reverses their characteristic local responses. Deactivation of both CaMKII and CaN seems dominated by Ca dissociation from the complex (versus Ca-CaM dissociation from the target). In summary, the different affinities of CaM for CaMKII and CaN determine their sensitivity to local Ca signals in cardiac myocytes.
Project description:We investigated the impact of bound calmodulin (CaM)-target compound structure on the affinity of calcium (Ca2+) by integrating coarse-grained models and all-atomistic simulations with nonequilibrium physics. We focused on binding between CaM and two specific targets, Ca2+/CaM-dependent protein kinase II (CaMKII) and neurogranin (Ng), as they both regulate CaM-dependent Ca2+ signaling pathways in neurons. It was shown experimentally that Ca2+/CaM (holoCaM) binds to the CaMKII peptide with overwhelmingly higher affinity than Ca2+-free CaM (apoCaM); the binding of CaMKII peptide to CaM in return increases the Ca2+ affinity for CaM. However, this reciprocal relation was not observed in the Ng peptide (Ng13-49), which binds to apoCaM or holoCaM with binding affinities of the same order of magnitude. Unlike the holoCaM-CaMKII peptide, whose structure can be determined by crystallography, the structural description of the apoCaM-Ng13-49 is unknown due to low binding affinity, therefore we computationally generated an ensemble of apoCaM-Ng13-49 structures by matching the changes in the chemical shifts of CaM upon Ng13-49 binding from nuclear magnetic resonance experiments. Next, we computed the changes in Ca2+ affinity for CaM with and without binding targets in atomistic models using Jarzynski's equality. We discovered the molecular underpinnings of lowered affinity of Ca2+ for CaM in the presence of Ng13-49 by showing that the N-terminal acidic region of Ng peptide pries open the ?-sheet structure between the Ca2+ binding loops particularly at C-domain of CaM, enabling Ca2+ release. In contrast, CaMKII peptide increases Ca2+ affinity for the C-domain of CaM by stabilizing the two Ca2+ binding loops. We speculate that the distinctive structural difference in the bound complexes of apoCaM-Ng13-49 and holoCaM-CaMKII delineates the importance of CaM's progressive mechanism of target binding on its Ca2+ binding affinities.
Project description:The Ca2+ sensor calmodulin (CaM) regulates cardiac ryanodine receptor (RyR2)-mediated Ca2+ release from the sarcoplasmic reticulum. CaM inhibits RyR2 in a Ca2+-dependent manner and aberrant CaM-dependent inhibition results in life-threatening cardiac arrhythmias. However, the molecular details of the CaM-RyR2 interaction remain unclear. Four CaM-binding domains (CaMBD1a, -1b, -2, and -3) in RyR2 have been proposed. Here, we investigated the Ca2+-dependent interactions between CaM and these CaMBDs by monitoring changes in the fluorescence anisotropy of carboxytetramethylrhodamine (TAMRA)-labeled CaMBD peptides during titration with CaM at a wide range of Ca2+ concentrations. We showed that CaM bound to all four CaMBDs with affinities that increased with Ca2+ concentration. CaM bound to CaMBD2 and -3 with high affinities across all Ca2+ concentrations tested, but bound to CaMBD1a and -1b only at Ca2+ concentrations above 0.2?µM. Binding experiments using individual CaM domains revealed that the CaM C-domain preferentially bound to CaMBD2, and the N-domain to CaMBD3. Moreover, the Ca2+ affinity of the CaM C-domain in complex with CaMBD2 or -3 was so high that these complexes are essentially Ca2+ saturated under resting Ca2+ conditions. Conversely, the N-domain senses Ca2+ exactly in the transition from resting to activating Ca2+ when complexed to either CaMBD2 or -3. Altogether, our results support a binding model where the CaM C-domain is anchored to RyR2 CaMBD2 and saturated with Ca2+ during Ca2+ oscillations, while the CaM N-domain functions as a dynamic Ca2+ sensor that can bridge noncontiguous regions of RyR2 or clamp down onto CaMBD2.
Project description:Calmodulin (CaM) associates with cardiac ryanodine receptor type-2 (RyR2) as an important regulator. Defective CaM-RyR2 interaction may occur in heart failure, cardiac hypertrophy, and catecholaminergic polymorphic ventricular tachycardia. However, the in situ binding properties for CaM-RyR2 are unknown.We sought to measure the in situ binding affinity and kinetics for CaM-RyR2 in normal and heart failure ventricular myocytes, estimate the percentage of Z-line-localized CaM that is RyR2-bound, and test cellular function of defective CaM-RyR2 interaction.Using fluorescence resonance energy transfer in permeabilized myocytes, we specifically resolved RyR2-bound CaM from other potential binding targets and measured CaM-RyR2 binding affinity in situ (Kd=10-20 nmol/L). Using RyR2(ADA/+) knock-in mice, in which half of the CaM-RyR2 binding is suppressed, we estimated that >90% of Z-line CaM is RyR2-bound. Functional tests indicated a higher propensity for Ca2+ wave production and stress-induced ventricular arrhythmia in RyR2(ADA/+) mice. In a post-myocardial infarction rat heart failure model, we detected a decrease in the CaM-RyR2 binding affinity (Kd?51 nmol/L; ?3-fold increase) and unaltered RyR2 affinity for the FK506-binding protein FKBP12.6 (Kd~0.8 nmol/L).CaM binds to RyR2 with high affinity in cardiac myocytes. Physiologically, CaM is bound to >70% of RyR2 monomers and inhibits sarcoplasmic reticulum Ca2+ release. RyR2 is the major binding site for CaM along the Z-line in cardiomyocytes, and dissociating CaM from RyR2 can cause severe ventricular arrhythmia. In heart failure, RyR2 shows decreased CaM affinity, but unaltered FKBP 12.6 affinity.
Project description:KEY POINTS:Ventricular arrhythmias are a major complication after myocardial infarction (MI), associated with sympathetic activation. The structurally heterogeneous peri-infarct zone is a known substrate, but the functional role of the myocytes is less well known. Recordings of monophasic action potentials in vivo reveal that the peri-infarct zone is a source of delayed afterdepolarizations (DADs) and has a high beat-to-beat variability of repolarization (BVR) during adrenergic stimulation (isoproterenol, ISO). Myocytes isolated from the peri-infarct region have more DADs and spontaneous action potentials, with spontaneous Ca2+ release, under ISO. These myocytes also have reduced repolarization reserve and increased BVR. Other properties of post-MI remodelling are present in both peri-infarct and remote myocytes. These data highlight the importance of altered myocyte adrenergic responses in the peri-infarct region as source and substrate of post-MI arrhythmias. ABSTRACT:Ventricular arrhythmias are a major early complication after myocardial infarction (MI). The heterogeneous peri-infarct zone forms a substrate for re-entry while arrhythmia initiation is often associated with sympathetic activation. We studied the mechanisms triggering these post-MI arrhythmias in vivo and their relation to regional myocyte remodelling. In pigs with chronic MI (6 weeks), in vivo monophasic action potentials were simultaneously recorded in the peri-infarct and remote regions during adrenergic stimulation with isoproterenol (isoprenaline; ISO). Sham animals served as controls. During infusion of ISO in vivo, the incidence of delayed afterdepolarizations (DADs) and beat-to-beat variability of repolarization (BVR) was higher in the peri-infarct than in the remote region. Myocytes isolated from the peri-infarct region, in comparison to myocytes from the remote region, had more DADs, associated with spontaneous Ca2+ release, and a higher incidence of spontaneous action potentials (APs) when exposed to ISO (9.99 ± 4.2 vs. 0.16 ± 0.05 APs/min, p = 0.004); these were suppressed by CaMKII inhibition. Peri-infarct myocytes also had reduced repolarization reserve and increased BVR (26 ± 10 ms vs. 9 ± 7 ms, P < 0.001), correlating with DAD activity. In contrast to these regional distinctions under ISO, alterations in Ca2+ handling at baseline and myocyte hypertrophy were present throughout the left ventricle (LV). Expression of some of the related genes was, however, different between the regions. In conclusion, altered myocyte adrenergic responses in the peri-infarct but not the remote region provide a source of triggered activity in vivo and of repolarization instability amplifying the substrate for re-entry. These findings stimulate further exploration of region-specific therapies targeting myocytes and autonomic modulation.
Project description:Fluxes of calcium (Ca2+) across cell membranes enable fast cellular responses. Calmodulin (CaM) senses local changes in Ca2+ concentration and relays the information to numerous interaction partners. The critical role of accurate Ca2+ signaling on cellular function is underscored by the fact that there are three independent CaM genes (CALM1-3) in the human genome. All three genes are functional and encode the exact same CaM protein. Moreover, CaM has a completely conserved amino acid sequence across all vertebrates. Given this degree of conservation, it was long thought that mutations in CaM were incompatible with life. It was therefore a big surprise when the first CaM mutations in humans were identified six years ago. Today, more than a dozen human CaM missense mutations have been described, all found in patients with severe cardiac arrhythmias. Biochemical studies have demonstrated differential effects on Ca2+ binding affinities for these CaM variants. Moreover, CaM regulation of central cardiac ion channels is impaired, including the voltage-gated Ca2+ channel, CaV1.2, and the sarcoplasmic reticulum Ca2+ release channel, ryanodine receptor isoform 2, RyR2. Currently, no non-cardiac phenotypes have been described for CaM variant carriers. However, sequencing of large human cohorts reveals a cumulative frequency of additional rare CaM mutations that raise the possibility of CaM variants not exclusively causing severe cardiac arrhythmias. Here, we provide an overview of the identified CaM variants and their known consequences for target regulation and cardiac disease phenotype. We discuss experimental data, patient genotypes and phenotypes as well as which questions remain open to understand this complexity.
Project description:Oxidative stress may contribute to cardiac ryanodine receptor (RyR2) dysfunction in heart failure (HF) and arrhythmias. Altered RyR2 domain-domain interaction (domain unzipping) and calmodulin (CaM) binding affinity are allosterically coupled indices of RyR2 conformation. In HF RyR2 exhibits reduced CaM binding, increased domain unzipping and greater SR Ca leak, and dantrolene can reverse these changes. However, effects of oxidative stress on RyR2 conformation and leak in myocytes are poorly understood. We used fluorescent CaM, FKBP12.6, and domain-peptide biosensor (F-DPc10) to measure, directly in cardiac myocytes, (1) RyR2 activation by hydrogen peroxide (H2O2)-induced oxidation, (2) RyR2 conformation change caused by oxidation, (3) CaM-RyR2 and FK506-binding protein (FKBP12.6)-RyR2 interaction upon oxidation, and (4) whether dantrolene affects 1-3. H2O2 was used to mimic oxidative stress. H2O2 significantly increased the frequency of Ca(2+) sparks and spontaneous Ca(2+) waves, and dantrolene almost completely blocked these effects. H2O2 pretreatment significantly reduced CaM-RyR2 binding, but had no effect on FKBP12.6-RyR2 binding. Dantrolene restored CaM-RyR2 binding but had no effect on intracellular and RyR2 oxidation levels. H2O2 also accelerated F-DPc10-RyR2 association while dantrolene slowed it. Thus, H2O2 causes conformational changes (sensed by CaM and DPc10 binding) associated with Ca leak, and dantrolene reverses these RyR2 effects. In conclusion, in cardiomyocytes, H2O2 treatment markedly reduces the CaM-RyR2 affinity, has no effect on FKBP12.6-RyR2 affinity, and causes domain unzipping. Dantrolene can correct domain unzipping, restore CaM-RyR2 affinity, and quiet pathological RyR2 channel gating. F-DPc10 and CaM are useful biosensors of a pathophysiological RyR2 state.
Project description:We have shown previously that the Ca2+-dependent inhibition of lens epithelial cell-to-cell communication is mediated in part by the direct association of calmodulin (CaM) with connexin43 (Cx43), the major connexin in these cells. We now show that elevation of [Ca2+](i) in HeLa cells transfected with the lens fiber cell gap junction protein sheep Cx44 also results in the inhibition of cell-to-cell dye transfer. A peptide comprising the putative CaM binding domain (aa 129-150) of the intracellular loop region of this connexin exhibited a high affinity, stoichiometric interaction with Ca2+-CaM. NMR studies indicate that the binding of Cx44 peptide to CaM reflects a classical embracing mode of interaction. The interaction is an exothermic event that is both enthalpically and entropically driven in which electrostatic interactions play an important role. The binding of the Cx44 peptide to CaM increases the CaM intradomain cooperativity and enhances the Ca2+-binding affinities of the C-domain of CaM more than twofold by slowing the rate of Ca2+ release from the complex. Our data suggest a common mechanism by which the Ca2+-dependent inhibition of the alpha-class of gap junction proteins is mediated by the direct association of an intracellular loop region of these proteins with Ca2+-CaM.
Project description:In cardiac myocytes Calmodulin (CaM) bound to the ryanodine receptor (RyR2) constitutes a large pool of total myocyte CaM, but the CaM-RyR2 affinity is reduced in pathological conditions. Knock-in mice expressing RyR2 unable to bind CaM also developed hypertrophy and early death. However, it is unknown whether CaM released from this RyR2-bound pool participates in pathological cardiac hypertrophy. We found that angiotensin II (AngII) or phenylephrine (PE) both cause CaM to dissociate from the RyR2 and translocate to the nucleus. To test whether this nuclear CaM accumulation depends on CaM released from RyR2, we enhanced CaM-RyR2 binding affinity (with dantrolene), or caused CaM dissociation from RyR2 (using suramin). Dantrolene dramatically reduced AngII- and PE-induced nuclear CaM accumulation. Conversely, suramin enhanced nuclear CaM accumulation. This is consistent with nuclear CaM accumulation coming largely from the CaM-RyR2 pool. CaM lacks a nuclear localization signal (NLS), but G-protein coupled receptor kinase 5 (GRK5) binds CaM, has a NLS and translocates like CaM in response to AngII or PE. Suramin also promoted GRK5 nuclear import, and caused nuclear export of histone deacetylase 5 (HDAC5). Dantrolene prevented these effects. After 2-8?weeks of pressure overload (TAC) CaM binding to RyR2 was reduced, nuclear CaM and GRK5 were both elevated and there was enhanced nuclear export of HDAC5. Stress (acute AngII or TAC) causes CaM dissociation from RyR2 and translocation to the nucleus with GRK5 with parallel HDAC5 nuclear export. Thus CaM dissociation from RyR2 may be an important step in driving pathological hypertrophic gene transcription.
Project description:BACKGROUND:Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic disorder caused by mutations in the cardiac ryanodine receptor RyR2 that increase diastolic calcium cation (Ca2+) leak from the sarcoplasmic reticulum (SR). Calmodulin (CaM) dissociation from RyR2 has been associated with diastolic Ca2+ leak in heart failure. OBJECTIVE:Determine whether the tetracaine-derivative compound EL20 inhibits abnormal Ca2+ release from RyR2 in a CPVT model and investigate the underlying mechanism of inhibition. METHODS:Spontaneous Ca2+ sparks in cardiomyocytes and inducible ventricular tachycardia were assessed in a CPVT mouse model, which is heterozygous for the R176Q mutation in RyR2 (R176Q/+ mice) in the presence of EL20 or vehicle. Single-channel studies using sheep cardiac SR or purified RyR2 reconstituted into proteoliposomes with and without exogenous CaM were used to assess mechanisms of inhibition. RESULTS:EL20 potently inhibits abnormal Ca2+ release in R176Q/+ myocytes (half-maximal inhibitory concentration = 35.4 nM) and diminishes arrhythmia in R176Q/+ mice. EL20 inhibition of single-channel activity of purified RyR2 occurs in a similar range as seen in R176Q/+ myocytes (half-maximal inhibitory concentration = 8.2 nM). Inhibition of single-channel activity for cardiac SR or purified RyR2 supplemented with 100-nM or 1-?M CaM shows a 200- to 1000-fold reduction in potency. CONCLUSION:This work provides a potential therapeutic mechanism for the development of antiarrhythmic compounds that inhibit leaky RyR2 resulting from CaM dissociation, which is often associated with failing hearts. Our data also suggest that CaM dissociation may contribute to the pathogenesis of arrhythmias with the CPVT-linked R176Q mutation.