Early cardiac hypertrophy in mice with impaired calmodulin regulation of cardiac muscle Ca release channel.
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ABSTRACT:
SUBMITTER: Yamaguchi N
PROVIDER: S-EPMC1847534 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Intracellular lysosomal membrane trafficking, including fusion and fission, is crucial for cellular homeostasis and normal cell function. Both fusion and fission of lysosomal membrane are accompanied by lysosomal Ca2+ release. We recently have demonstrated that the lysosomal Ca2+ release channel P2X4 regulates lysosome fusion through a calmodulin (CaM)-dependent mechanism. However, the molecular mechanism underlying lysosome fission remains uncertain. In this study, we report that enlarged lysosomes/vacuoles induced by either vacuolin-1 or P2X4 activation are suppressed by up-regulating the lysosomal Ca2+ release channel transient receptor potential mucolipin 1 (TRPML1) but not the lysosomal Na+ release channel two-pore channel 2 (TPC2). Activation of TRPML1 facilitated the recovery of enlarged lysosomes/vacuoles. Moreover, the effects of TRPML1 on lysosome/vacuole size regulation were eliminated by Ca2+ chelation, suggesting a requirement for TRPML1-mediated Ca2+ release. We further demonstrate that the prototypical Ca2+ sensor CaM is required for the regulation of lysosome/vacuole size by TRPML1, suggesting that TRPML1 may promote lysosome fission by activating CaM. Given that lysosome fission is implicated in both lysosome biogenesis and reformation, our findings suggest that TRPML1 may function as a key lysosomal Ca2+ channel controlling both lysosome biogenesis and reformation.
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Project description:Antagonists of L-type Ca²⁺ channels (LTCCs) have been used to treat human cardiovascular diseases for decades. However, these inhibitors can have untoward effects in patients with heart failure, and their overall therapeutic profile remains nebulous given differential effects in the vasculature when compared with those in cardiomyocytes. To investigate this issue, we examined mice heterozygous for the gene encoding the pore-forming subunit of LTCC (calcium channel, voltage-dependent, L type, α1C subunit [Cacna1c mice; referred to herein as α1C⁻/⁺ mice]) and mice in which this gene was loxP targeted to achieve graded heart-specific gene deletion (termed herein α1C-loxP mice). Adult cardiomyocytes from the hearts of α1C⁻/⁺ mice at 10 weeks of age showed a decrease in LTCC current and a modest decrease in cardiac function, which we initially hypothesized would be cardioprotective. However, α1C⁻/⁺ mice subjected to pressure overload stimulation, isoproterenol infusion, and swimming showed greater cardiac hypertrophy, greater reductions in ventricular performance, and greater ventricular dilation than α1C⁺/⁺ controls. The same detrimental effects were observed in α1C-loxP animals with a cardiomyocyte-specific deletion of one allele. More severe reductions in α1C protein levels with combinatorial deleted alleles produced spontaneous cardiac hypertrophy before 3 months of age, with early adulthood lethality. Mechanistically, our data suggest that a reduction in LTCC current leads to neuroendocrine stress, with sensitized and leaky sarcoplasmic reticulum Ca²⁺ release as a compensatory mechanism to preserve contractility. This state results in calcineurin/nuclear factor of activated T cells signaling that promotes hypertrophy and disease.
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Project description:AimsCalmodulin (CaM) is well known to modulate the channel function of the cardiac ryanodine receptor (RyR2). However, the possible role of CaM on the aberrant Ca(2+) release in diseased hearts remains unclear. In this study, we investigated the state of RyR2-bound CaM and channel dysfunctions in pacing-induced failing hearts.Methods and resultsThe characteristics of CaM binding to RyR2 and the role of CaM on the aberrant Ca(2+) release were assessed in normal and failing canine hearts. The affinity of CaM binding to RyR2 was lower in failing sarcoplasmic reticulum (SR) than in normal SR. Addition of FK506, which dissociates FKBP12.6 from RyR2, to normal SR reduced the CaM-binding affinity. Dantrolene restored a normal level of the CaM-binding affinity in either FK506-treated (normal) SR or failing SR, suggesting that the defective inter-domain interaction between the N-terminal domain and the central domain of RyR2 (the therapeutic target of dantrolene) is involved in the reduction of the CaM-binding affinity in failing hearts. In saponin-permeabilized cardiomyocytes, the frequency of spontaneous Ca(2+) sparks was much more increased in failing cardiomyocytes than in normal cardiomyocytes, whereas the addition of a high concentration of CaM attenuated the aberrant increase of Ca(2+) sparks.ConclusionThe defective inter-domain interaction between N-terminal and central domains within RyR2 reduces the binding affinity of CaM to RyR2, thereby causing the spontaneous Ca(2+) release events in failing hearts. Correction of the defective CaM binding may be a new strategy to protect against the aberrant Ca(2+) release in heart failure.
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Project description:Calmodulin (CaM) is proposed to modulate activity of the skeletal muscle sarcoplasmic reticulum (SR) calcium release channel (ryanodine receptor, RyR1 isoform) via a mechanism dependent on the conformation of RyR1-bound CaM. However, the correlation between CaM structure and functional regulation of RyR in physiologically relevant conditions is largely unknown. Here, we have used time-resolved fluorescence resonance energy transfer (TR-FRET) to study structural changes in CaM that may play a role in the regulation of RyR1. We covalently labeled each lobe of CaM (N and C) with fluorescent probes and used intramolecular TR-FRET to assess interlobe distances when CaM is bound to RyR1 in SR membranes, purified RyR1, or a peptide corresponding to the CaM-binding domain of RyR (RyRp). TR-FRET resolved an equilibrium between two distinct structural states (conformations) of CaM, each characterized by an interlobe distance and Gaussian distribution width (disorder). In isolated CaM, at low Ca2+, the two conformations of CaM are resolved, centered at 5 nm (closed) and 7 nm (open). At high Ca2+, the equilibrium shifts to favor the open conformation. In the presence of RyRp at high Ca2+, the closed conformation shifts to a more compact conformation and is the major component. When CaM is bound to full-length RyR1, either purified or in SR membranes, strikingly different results were obtained: 1) the two conformations are resolved and more ordered, 2) the open state is the major component, and 3) Ca2+ stabilized the closed conformation by a factor of two. We conclude that the Ca2+-dependent structural distribution of CaM bound to RyR1 is distinct from that of CaM bound to RyRp. We propose that the function of RyR1 is tuned to the Ca2+-dependent structural dynamics of bound CaM.
| S-EPMC7063434 | biostudies-literature
Project description:Calmodulin (CaM) plays a critical role in intracellular signaling and regulation of Ca2+-dependent proteins and ion channels. Mutations in CaM cause life-threatening cardiac arrhythmias. Among the known CaM targets, small-conductance Ca2+-activated K+ (SK) channels are unique, since they are gated solely by beat-to-beat changes in intracellular Ca2+. However, the molecular mechanisms of how CaM mutations may affect the function of SK channels remain incompletely understood. To address the structural and functional effects of these mutations, we introduced prototypical human CaM mutations in human induced pluripotent stem cell-derived cardiomyocyte-like cells (hiPSC-CMs). Using structural modeling and molecular dynamics simulation, we demonstrate that human calmodulinopathy-associated CaM mutations disrupt cardiac SK channel function via distinct mechanisms. CaMD96V and CaMD130G mutants reduce SK currents through a dominant-negative fashion. By contrast, specific mutations replacing phenylalanine with leucine result in conformational changes that affect helix packing in the C-lobe, which disengage the interactions between apo-CaM and the CaM-binding domain of SK channels. Distinct mutant CaMs may result in a significant reduction in the activation of the SK channels, leading to a decrease in the key Ca2+-dependent repolarization currents these channels mediate. The findings in this study may be generalizable to other interactions of mutant CaMs with Ca2+-dependent proteins within cardiac myocytes.
| S-EPMC7681919 | biostudies-literature
Project description:RationaleCaMKII (Ca2+-Calmodulin dependent protein kinase) δC activation is implicated in pathological progression of heart failure (HF) and CaMKIIδC transgenic mice rapidly develop HF and arrhythmias. However, little is known about early spatio-temporal Ca2+ handling and CaMKII activation in hypertrophy and HF.ObjectiveTo measure time- and location-dependent activation of CaMKIIδC signaling in adult ventricular cardiomyocytes, during transaortic constriction (TAC) and in CaMKIIδC transgenic mice.Methods and resultsWe used human tissue from nonfailing and HF hearts, 4 mouse lines: wild-type, KO (CaMKIIδ-knockout), CaMKIIδC transgenic in wild-type (TG), or KO background, and wild-type mice exposed to TAC. Confocal imaging and biochemistry revealed disproportional CaMKIIδC activation and accumulation in nuclear and perinuclear versus cytosolic regions at 5 days post-TAC. This CaMKIIδ activation caused a compensatory increase in sarcoplasmic reticulum Ca2+ content, Ca2+ transient amplitude, and [Ca2+] decline rates, with reduced phospholamban expression, all of which were most prominent near and in the nucleus. These early adaptive effects in TAC were entirely mimicked in young CaMKIIδ TG mice (6-8 weeks) where no overt cardiac dysfunction was present. The (peri)nuclear CaMKII accumulation also correlated with enhanced HDAC4 (histone deacetylase) nuclear export, creating a microdomain for transcriptional regulation. At longer times both TAC and TG mice progressed to overt HF (at 45 days and 11-13 weeks, respectively), during which time the compensatory Ca2+ transient effects reversed, but further increases in nuclear and time-averaged [Ca2+] and CaMKII activation occurred. CaMKIIδ TG mice lacking δB exhibited more severe HF, eccentric myocyte growth, and nuclear changes. Patient HF samples also showed greatly increased CaMKIIδ expression, especially for CaMKIIδC in nuclear fractions.ConclusionsWe conclude that in early TAC perinuclear CaMKIIδC activation promotes adaptive increases in myocyte Ca2+ transients and nuclear transcriptional responses but that chronic progression of this nuclear Ca2+-CaMKIIδC axis contributes to eccentric hypertrophy and HF.
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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.
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Project description:Cardiac muscle adapts to hemodynamic stress by altering myocyte size and function, resulting in cardiac hypertrophy. Alteration in myocyte calcium homeostasis is known to be an initial signal in cardiac hypertrophy signaling. Transient receptor potential melastatin 4 protein (TRPM4) is a calcium-activated non-selective cation channel, which plays a role in regulating calcium influx and calcium-dependent cell functions in many cell types including cardiomyocytes. Selective deletion of TRPM4 from the heart muscle in mice resulted in an increased hypertrophic growth after chronic angiotensin (AngII) treatment, compared to WT mice. The enhanced hypertrophic response was also traceable by the increased expression of hypertrophy-related genes like Rcan1, ANP, and α-Actin. Intracellular calcium measurements on isolated ventricular myocytes showed significantly increased store-operated calcium entry upon AngII treatment in myocytes lacking the TRPM4 channel. Elevated intracellular calcium is a key factor in the development of pathological cardiac hypertrophy, leading to the activation of intracellular signaling pathways. In agreement with this, we observed significantly higher Rcan1 mRNA level, calcineurin enzyme activity and protein level in lysates from TRPM4-deficient mice heart compared to WT after AngII treatment. Collectively, these observations are consistent with a model in which TRPM4 is a regulator of calcium homeostasis in cardiomyocytes after AngII stimulation. TRPM4 contributes to the regulation of driving force for store-operated calcium entry and thereby the activation of the calcineurin-NFAT pathway and the development of pathological hypertrophy.
| S-EPMC4456993 | biostudies-literature
Project description:AimsAlthough aberrant Ca(2+) release (i.e. Ca(2+) 'leak') from the sarcoplasmic reticulum (SR) through cardiac ryanodine receptors (RyR2) is linked to heart failure (HF), it remains unknown whether and under what conditions SR-derived Ca(2+) can actually cause HF. We tested the hypothesis that combining dysregulated RyR2 function with facilitated Ca(2+) uptake into SR will exacerbate abnormal SR Ca(2+) release and induce HF. We also examined the mechanisms for these alterations.Methods and resultsWe crossbred mice deficient in expression of cardiac calsequestrin (CASQ2) with mice overexpressing the skeletal muscle isoform of SR Ca(2+)ATPase (SERCA1a). The new double-mutant strains displayed early mortality, congestive HF with left ventricular dilated hypertrophy, and decreased ejection fraction. Intact right ventricular muscle preparations from double-mutant mice preserved normal systolic contractile force but were susceptible to spontaneous contractions. Double-mutant cardiomyocytes while preserving normal amplitude of systolic Ca(2+) transients displayed marked disturbances in diastolic Ca(2+) handling in the form of multiple, periodic Ca(2+) waves and wavelets. Dysregulated myocyte Ca(2+) handling and structural and functional cardiac pathology in double-mutant mice were associated with increased rate of apoptotic cell death. Qualitatively similar results were obtained in a hybrid strain created by crossing CASQ2 knockout mice with mice deficient in phospholamban.ConclusionWe demonstrate that enhanced SR Ca(2+) uptake combined with dysregulated RyR2s results in sustained diastolic Ca(2+) release causing apoptosis, dilated cardiomyopathy, and early mortality. Our data also suggest that up-regulation of SERCA activity must be advocated with caution as a therapy for HF in the context of abnormal RyR2 function.
| S-EPMC3633154 | biostudies-literature
Project description:Mammalian brain possesses ryanodine-sensitive Ca2+ channels, which in muscle cells mediate rapid Ca2+ release from intracellular stores during excitation-contraction coupling. Analysis of bovine brain ryanodine receptor (RyR) channels suggests specific expression of the cardiac-muscle RyR isoform in mammalian brain. Localization using cardiac-muscle RyR-specific antibodies and antisense RNA revealed that brain RyRs were present in dendrites, cell bodies and terminals of rat forebrain, and highly enriched in the hippocampus. Activity of skeletal-muscle RyR channels is coupled to sarcolemmal voltage sensors, in contrast with cardiac-muscle RyR channels, which are known to be Ca(2+)-induced Ca(2+)-release channels. Thus Ca(2+)-induced Ca2+ release from intracellular stores mediated by brain RyR channels may be a major Ca(2+)-signalling pathway in specific regions of mammalian brain, and hence may play a fundamental role in neuronal Ca2+ homoeostasis.
| S-EPMC1132046 | biostudies-other