Cytoplasmic CaMKIIδ-B prevents myocardial recovery in heart failure

Figures

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  Fig. 1.. Myocardial recovery in heart failure is associated with differential alternative splicing. (A-B) Left ventricular ejection fraction (LVEF) (A) and LV end diastolic dimension (LVEDD) (B) in pre- and post-LVAD in responders (R) and non-responders (NR); n = 10 R, 9 NR; paired two-tailed t-test. (C-D) GSEA gene ontology (GO) Biological Process (BP) analysis of bulk RNA-seq data for genes with expression changes pre- to post-LVAD that were positively (C) or negatively (D) associated with a favorable response to LVAD. (E) Volcano plot displaying quantitative proteomics results from post-LVAD responder (R) and non-responder (NR) myocardium; n = 9/group. (F) Gene ontology (GO) Biological Process pathway enrichment analysis of the significantly upregulated proteins in Responders. (G) Heat map displaying the significantly differentially expressed RNA splicing proteins between post-LVAD R and NR. (H) Venn diagram displaying the number of local splicing variations identified between HF (n = 19) and control (n = 5) by RNA-seq and the differential alternative splicing responses post-LVAD in R (n = 10) and NR (n = 9) patients. (I) TTN exon 242 alternative splicing map and quantification between…

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  Fig. 2.. Increased CAMK2D exon 14 inclusion in heart failure is reversed in recovery. (A) Graphical representation of cardiac CAMK2D isoforms and their respective variable region (exons 13–17) alternative splicing; NLS = nuclear localization signal. (B) CAMK2D exon 14 proportion spliced in (PSI) in pre- and post-LVAD responders and non-responders from the RNA-seq data; two-way ANOVA with Tukey’s post-hoc test. (C-D) Linear regression analysis of exon 14 PSI (post-LVAD) vs. absolute change in LVEF (B) and % change in LVEDD (C) on LVAD therapy; confidence interval = 95%. (E) Representative RT-PCR acrylamide gel for CAMK2D cardiac isoforms and PGK1 housekeeping gene in non-failing controls, HF, and post-LVAD R and NR; BP = base-pairs. (F) Proportion CAMK2D isoform expression from the RT-PCR; n = 6 Control, 10 HF and LVAD R, 9 HF and LVAD NR. (G) Quantitative RT-PCR analysis of CAMK2D-B in Control, HF (pre-LVAD), and post-LVAD normalized to the housekeeping gene PGK1 and plotted as a fold-change vs. non-failing control expression; n = 10 R, 9 NR; one-way ANOVA with Tukey’s post-hoc test.

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  Fig. 3.. CaMKIIδ-B S332-S334 phosphorylation predicts poor functional recovery. (A) Volcano plot displaying phospho-proteomics results from post-LVAD responder (R) and non-responder (NR) myocardium; n = 9/group. (B) CaMKIIδ-B S332, S333, and S334 phosphorylation between R and NR post-LVAD; two-tailed t-test; *p < 0.05, **p < 0.01, ****p < 0.0001. (C) Representative western blots for total CaMKIIδ, ox-M281/282, p-T287, and p-S332 in non-failing control, pre-LVAD (HF) R and NR, and post-LVAD R and NR. (D-F) Ox-M281/282 CaMKIIδ (D), p-T287 CaMKIIδ (E), and p-S332 CaMKIIδ (F) normalized to total protein; n = 6 Control, 10 HF and LVAD R, 9 HF and LVAD NR; one-way ANOVA with Tukey’s post-hoc test. (G) Linear regression analysis of post-LVAD S332 phosphorylation vs. absolute change in LVEF on LVAD therapy; y-axis line at 15 (minimum value to assign positive functional response), x-axis line at 1.5-fold increase in p-S332 vs. non-failing controls; confidence interval = 95%. Data are presented as the mean ± SEM.

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  Fig. 4.. Phosphorylation at S332-S334 prevents autoactivation-dependent CaMKIIδ-B nuclear translocation. (A) Representative images for NRVMs 24-hours post-transduction with GFP-CaMKIIδ-B adenoviruses ± PE (20 μM); BSSS = wildtype, BAAA = Serine 332–334 mutated to Alanine (Phospho-null), BDDD = Serine 332–334 mutated to Aspartic Acid (Phospho-mimetic). (B) Nuclear GFP normalized to whole-cell GFP; Vehicle: n = 75 BSSS, 82 BAAA, and 72 BDDD. PE: n = 57 BSSS + PE, 72 BAAA, 91 BDDD from three biological replicates; two-way ANOVA with Tukey’s post-hoc test. (C) Representative western blot for GFP-BAAA in the nuclear and cytosolic fractions of NRVMs treated with vehicle or PE. (D) Nuclear GFP-BAAA normalized to Histone H3; n = 4/group; two-tailed t-test. (E) Representative western blots for M281/282 oxidized and T287 phosphorylated GFP-CaMKIIδ-B in adenovirus-transduced NRVMs ± PE. (F) p-T287 normalized to GAPDH; n = 6/group; two-way ANOVA with Tukey’s post-hoc test. (G) Representative images for NRVMs 24-hours post-transduction with BAAA or BAAA-T287A/D mutants. (H) Nuclear GFP normalized to whole-cell GFP; Vehicle: n = 16 BAAA, 82 T287A, and 68 T287D. PE: n = 66 BAAA, 91 T287A, and 117 T287D…

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  Fig. 5.. Cytoplasmic CaMKIIδ-B remodels the cardiomyocyte phospho-proteome and impairs contractility. (A) Heat map depicting the differentially expressed phospho-peptides in NRVMs transduced with empty vector (E), BAAA, or BDDD ± PE; p-value < 0.01, log2 fold-change < −0.3, > 0.3. (B) Volcano plot displaying differential phospho-peptide expression in NRVMs transduced with BAAA or BDDD. (C) Volcano plot displaying differential phosphor-peptide expression in NRVMs transduced with BAAA or BDDD and treated with PE. (D-E) Reactome Pathway over-enrichment for the differentially expressed phospho-peptides in BAAA + PE (D) versus BDDD + PE (E). (F) Representative images of EHTs 24 hours post-transduction with Empty vector, GFP-BAAA, or GFP-BDDD adenoviruses; scale bar = 1 mm. (G) Representative calcium transient traces for EHTs with and without treatment with the dual α and β adrenergic agonists PE, 50 μM, and isoproterenol (ISO), 2 μM. Traces are plotted as % change in fluorescence (ΔF) over time. (H) Mean calcium transient amplitude; Vehicle: n = 41 Empty vector, 43 BAAA, 41 BDDD EHTs from three biological replicates. ISO/PE: n = 14 EHTs/group from two biological replicates; two-way ANOVA with…