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MCUb upregulation in the type 2 diabetic heart impacts cardiac function by altering mitochondrial metabolic fuel flux and the cardiac myocyte phosphoproteome

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE145294 | GEO | 2022/12/15

REPOSITORIES: GEO

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Project description:Background: Phospholamban (PLN) is a tail-anchored sarco-endoplasmic reticulum (SER) membrane protein that regulates cardiac contraction/relaxation by reversibly inhibiting the SERCA2a Ca2+ 36 -reuptake pump. The R14Δ-PLN mutation causes severe cardiomyopathy that is resistant to conventional treatment. Multi-protein complexes and higher-order supercomplexes, such as intercalated disk components and SER Ca+2 -cycling domains, underlie many critical cardiac functions, a subset of which may be disrupted by R14∆-PLN. Complexome profiling (CP) is a systems biology approach to characterize high molecular weight (MW) assemblies in biological samples. Most current CP data analysis strategies rely on external protein42 complex databases that are predominantly curated from immortalized or cancerous cell lines; thus, assemblies unique to specialized cell types such as cardiomyocytes, as well as higher-order supercomplexes that may have fluid stoichiometry/composition are significantly underrepresented. The requirement for computer programming expertise also presents a barrier to generalized adoption. Methods: We developed and applied a novel CP workflow to identify protein-complexes/supercomplexes disrupted in presymptomatic 28wk-old R14Δ/+ knockin mice hearts. Cardiac tissues were homogenized under non-denaturing conditions, fractionated by size-exclusion chromatography (SEC) and subjected to quantitative data-independent acquisition mass spectrometry (DIA-MS)-based proteomics analysis. As expected, traditional database-dependent analysis offered only limited insights due to underrepresentation of cardiac-specific assemblies and higher-order supercomplexes. We thus developed a novel data analysis strategy (PERCOM) that does not rely upon external protein-complex databases and can, furthermore, be implemented on widely available spreadsheet software. Results: SEC-DIA-MS coupled with PERCOM analysis identified 296 proteins with disrupted MW-distribution profiles. Hits were significantly enriched for mitochondrial and intercalated disk (ICD) components. Mitochondrial alterations were confirmed at the level of global protein expression and metabolic function. Disruption of mitochondrial and ICD supercomplexes was confirmed in mice as young as 9wks of age. Conclusion: We present a novel CP workflow that is highly suited to the analysis of very high MW protein-complexes and supercomplexes in specialized tissue types. It includes a data analysis strategy that removes a computer programming resource barrier to general application. Our findings are among the first to show altered mitochondrial function as a hallmark of early-stage R14Δ-PLN, and identify disruption of the intercalated disk as a novel potential disease pathway. These molecular-level alterations target key machinery underlying cardiac energy generation and electromechanical connectivity, and may significantly contribute to the R14∆-PLN pathogenesis.

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MCUb upregulation in the type 2 diabetic heart impacts cardiac function by altering mitochondrial metabolic fuel flux and the cardiac myocyte phosphoproteome

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