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Sustained L-type Calcium Channel Blockade Alters Gene Regulation in the Adult Mouse Ventricle

ABSTRACT: Objective: L-type calcium channels (LTCC) homeostatically regulate calcium on a beat by beat basis, but also provide Ca that over long time scales may contribute to transcriptional regulation. We previously showed that sustained LTCC blockade (CCB) elicits LTCC remodeling in ventricular cardiac myocytes (CM). Here we hypothesize that sustained CCB has broad effects on the expression of genes involved in calcium handling. Methods and Results: Therefore, we subjected adult mice to sustained CCB for 24 hours and performed gene expression profiling. In comparison to vehicle-only control animals, 231 genes were up-regulated, and 111 genes were down-regulated by sustained LTCC blockade (p <0.01). Gene ontology analysis suggested that the CaMKIIdelta signaling pathway was up-regulated in these cells. Unexpectedly, phosphorylation of phospholamban (PLN) at threonine17 (Thr17), an index of CaMKIIdelta activity, was not changed by sustained CCB; however, the degree of phosphorylation of the neighboring PLN-Ser16 substrate site for PKA was significantly reduced by sustained CCB compared to control. Gene expression profiling suggested no change in PKA, but it showed that protein phosphatase 2A (PP2A) mRNA increased, and immunoblots demonstrated that PP2Ac-alpha protein was significantly increased by sustained CCB. Consistent with elevated PP2Ac-alpha protein expression LTCC exhibited decreased phosphorylation of the C-terminal Ser1928 PKA substrate site. Conclusions: We conclude that sustained CCB elicits a spectrum of transcriptional events, including compensatory up-regulation of LTCC and PP2Ac-alpha. Although this study is restricted to mouse, these results suggest the new hypothesis that clinically-relevant sustained LTCC blockade in humans results in changes in gene regulation in the heart. Keywords: L-type calcium channel, calcium channel blockade, verapamil

ORGANISM(S): Mus musculus

PROVIDER: GSE8367 | GEO | 2009/12/31

SECONDARY ACCESSION(S): PRJNA101393

REPOSITORIES: GEO

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Project description:Objective: L-type calcium channels (LTCC) homeostatically regulate calcium on a beat by beat basis, but also provide Ca that over long time scales may contribute to transcriptional regulation. We previously showed that sustained LTCC blockade (CCB) elicits LTCC remodeling in ventricular cardiac myocytes (CM). Here we hypothesize that sustained CCB has broad effects on the expression of genes involved in calcium handling. Methods and Results: Therefore, we subjected adult mice to sustained CCB for 24 hours and performed gene expression profiling. In comparison to vehicle-only control animals, 231 genes were up-regulated, and 111 genes were down-regulated by sustained LTCC blockade (p <0.01). Gene ontology analysis suggested that the CaMKIIdelta signaling pathway was up-regulated in these cells. Unexpectedly, phosphorylation of phospholamban (PLN) at threonine17 (Thr17), an index of CaMKIIdelta activity, was not changed by sustained CCB; however, the degree of phosphorylation of the neighboring PLN-Ser16 substrate site for PKA was significantly reduced by sustained CCB compared to control. Gene expression profiling suggested no change in PKA, but it showed that protein phosphatase 2A (PP2A) mRNA increased, and immunoblots demonstrated that PP2Ac-alpha protein was significantly increased by sustained CCB. Consistent with elevated PP2Ac-alpha protein expression LTCC exhibited decreased phosphorylation of the C-terminal Ser1928 PKA substrate site. Conclusions: We conclude that sustained CCB elicits a spectrum of transcriptional events, including compensatory up-regulation of LTCC and PP2Ac-alpha. Although this study is restricted to mouse, these results suggest the new hypothesis that clinically-relevant sustained LTCC blockade in humans results in changes in gene regulation in the heart. Keywords: L-type calcium channel, calcium channel blockade, verapamil Female and male ICR mice (12-14 weeks age) weighing between 25 and 30 grams were anesthetized with a ketamine/xylazine mixture ( i.p.) allowing the subcutaneous implantation of miniosmotic pumps (Alzet, model 2001). The pumps were filled with either verapamil or vehicle (0.02% ascorbic acid). Control animals carried mini-pumps with vehicle and control animals were investigated in parallel with each set of experimental animals. Mini pumps delivered verapamil at 3.6 mg/kg/day for 24 (RNA)-48 (protein) hours. After treatment animals were anesthetized and weighed. Hearts were excised, rinsed, blotted dry, weighed, and then frozen on dry ice and the stored at -80oC until studied. Animals were anesthetized and euthanized according to animal protocols approved by the University of Kentucky Institutional Animal Care and Use Committee. This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication NO. 85-23, revised 1996). Left ventricular free wall from female mice was rapidly excised and either snap frozen at -80oC or used immediately for RNA isolation. Three VER treated mice and 3 vehicle treated mice were used to generate RNA for microarray. Total RNA was isolated using the RNAqueous -4PCR kit (Ambion) and quantitated spectrophotometrically at 260nm. Contaminating genomic DNA was eliminated by DNase treatment (Ambion). RNA quality was assessed using the Agilent 2100 Bioanalyzer. Microarray data was obtained using the Affymetrix 430 V2 GeneChip (representing 45,101 probe sets), in accordance with the manufacturer’s specifications.

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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Sustained L-type Calcium Channel Blockade Alters Gene Regulation in the Adult Mouse Ventricle

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