Dataset Information

Molecular basis of b-adrenergic regulation of cardiac calcium current

ABSTRACT: Increased cardiac contractility during fight-or-flight response is caused by b-adrenergic augmentation of CaV1.2 channels. It is assumed that this iconic regulation involves phosphorylation of CaV1.2 a1/b-subunits. In transgenic murine hearts expressing fully PKA phosphorylation-deficient mutant a1C/b, this regulation persists, however, suggesting involvement of extra-channel factors. We here show that PKA up-regulates cardiac CaV1.2 channels by phosphorylating the small G-protein Rad, relieving constitutive inhibition of CaV1.2. Peroxidase-catalyzed labeling in mice hearts expressing ascorbate peroxidase conjugated-a1C or b2b with multiplexed quantitative proteomics allowed tracking of thousands of proteins in proximity of CaV1.2.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Heart, Cardiac Muscle Cell

DISEASE(S): Cardiovascular System Disease,Cervix Carcinoma

SUBMITTER: Marian Kalocsay

LAB HEAD: Marian Kalocsay

PROVIDER: PXD014499 | Pride | 2019-11-19

REPOSITORIES: Pride

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Mechanism of adrenergic CaV1.2 stimulation revealed by proximity proteomics.

Liu Guoxia G Papa Arianne A Katchman Alexander N AN Zakharov Sergey I SI Roybal Daniel D Hennessey Jessica A JA Kushner Jared J Yang Lin L Chen Bi-Xing BX Kushnir Alexander A Dangas Katerina K Gygi Steven P SP Pitt Geoffrey S GS Colecraft Henry M HM Ben-Johny Manu M Kalocsay Marian M Marx Steven O SO

Nature 20200122 7792

Increased cardiac contractility during the fight-or-flight response is caused by β-adrenergic augmentation of CaV1.2 voltage-gated calcium channels1-4. However, this augmentation persists in transgenic murine hearts expressing mutant CaV1.2 α1C and β subunits that can no longer be phosphorylated by protein kinase A-an essential downstream mediator of β-adrenergic signalling-suggesting that non-channel factors are also required. Here we identify the mec ...[more]

PMID: 31969708

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Project description:Voltage gated calcium channels play a central role in regulating the electrical and biochemical properties of neurons and muscle cells. Cells coordinate the expression of voltage gated calcium channels with the expression of other proteins that regulate membrane potential and calcium homeostasis. We report that the C-terminus of CaV 1.2, an L-type calcium channel (LTC) contains a C-terminal fragment that translocates to the nucleus and regulates transcription. This calcium channel associated transcription factor (CCAT) associates with transcriptional co-regulators such as p54nrb/NonO and binds to endogenous promoters. CCAT regulates the expression of gap junctions, sodium calcium exchangers, NMDA receptors, potassium channels and other proteins that regulate neuronal signaling. Electrical activity and developmental processes regulate the nuclear localization of CCAT, suggesting that the CCAT integrates information about the number of LTCs with information about the developmental history and electrical activity of a cell. These findings provide the first evidence that voltage gated calcium channels can directly activate transcription and suggest a novel mechanism linking voltage gated channels to the function and differentiation of excitable cells. Experiment Overall Design: The goal of these experiments was to identify the genes that are regulated by CCAT, a novel transcription factor derived from the C-terminus of CaV1.2. Neuro2A neuroblastoma cells were transfected with the last 503 AA of CaV1.2 which is full length CCAT (CCAT FL) or with the last 280 AA of CaV1.2, a form of CCAT that lacks the transcriptional activation domain (CCAT DTA). The mRNA from either CCAT FL or CCAT DTA expressing cells was hybridized to Agilent mouse genome microarrays along with mRNA from untransfected neuro2A cells (CCAT FL or DTA A series). Subsequent investigation revealed that transfection with the PA1 plasmid by itself increases the expression of some genes. To control for this effect we compared Neuro2A cells transfected with full length CCAT (in PA1) with Neuro2A cells transfectected with PA1 alone (CCAT FL B series). The microarray data was analyzed with the Rossetta Luminator gene expression data analysis system.

Project description:Mitochondrial Ca2+ (mCa2+) plays an important role in regulating cardiac metabolism and physiology. However, its contribution to metabolic and functional defects in the hearts of type 2 diabetic mice (T2D) is not well understood. Ca2+ is imported into mitochondria by the mitochondrial calcium uniporter complex (MCUC) which is a highly selective multimeric Ca2+ ion channel composed of pore-forming, scaffold, and regulatory subunits. Here, we describe new mechanisms involving the MCUC inhibitory subunit MCUb and its role in determining cardiac dysfunction by linking cardiac mitochondrial metabolism and contractile function. Hearts from T2D mice were analyzed 5 months following a single streptozotocin (STZ) injection (75 mg/kg) and high fat diet (HFD) feeding, and compared to control hearts from mice fed normal chow. Transcriptional and proteomic profiling were employed to characterize these hearts. Regulation of MCUb gene expression was investigated using a recombinant mutant inactive Cas9 (dCas9)-based gene promoter pull down approach coupled with mass spectrometry (MS). A dominant negative MCUb (MCUb W246R/V251E=dnMCUb) was engineered. dnMCUb was biochemically characterized in vitro, tested in neonatal cardiac myocytes exposed to culture media mimicking the diabetic milieu, and expressed in type 2 diabetic mice for 1 month. The impact of dnMCUb on the cardiac physiology was then investigated using omics as well as functional approaches aimed to highlight metabolic and contractile features. RNA-seq and proteomics analysis revealed that T2D hearts relied almost exclusively on fatty acid oxidative metabolism. In parallel we found a significant increase in MCUb mRNA and protein in T2D hearts, which was associated with decreased mCa2+ import rates. MS analysis of proteins co-precipitated with dCas9 revealed the presence of NCOR2 in control but not in T2D MCUb gene promoters. Downregulation of NCOR2 in isolated cardiac myocytes from control mice recapitulated the increase in MCUb gene expression. The dnMCUb mutant increased MCUC Ca2+ affinity by improving the architecture of the complex. Furthermore, when dnMCUb was introduced to cardiac myocytes and T2D mice we observed increased mitochondrial glucose oxidation, mitochondrial ATP production, and enhanced cardiac function. These were linked to the complete restoration of phospholamban (PLN) phosphorylation via Protein Kinase A (PKA) as revealed by both MS analysis of the phosphoproteome and studies dedicated on PLN only followed by PKA pharmacological inhibition. We detail a new pathological axis in which T2D induces global metabolic adaptations and inflexibility in part due to impaired mCa2+ transport into the mitochondrial matrix, resulting from MCUb overexpression. Moreover, we describe a mCa2+-dependent regulation of cytosolic Ca2+ (cCa2+) trafficking in which mCa2+ regulates the activity of SERCA2a via PKA-dependent phosphorylation of PLN.

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Dataset Information

Molecular basis of b-adrenergic regulation of cardiac calcium current

Dataset's files

Publications

Mechanism of adrenergic Ca<sub>V</sub>1.2 stimulation revealed by proximity proteomics.

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