Fig 2: Results of protein analyses. Representative examples of channel proteins (upper subpanels) and summarized results of channel proteins (lower subpanels) (n = 6, Student’s t-test for all comparisons). Non-phosphorylated connexin43 (NP-Cx43), calmodulin-dependent protein kinase II (CaMKII), and pThr287-CaMKII (CaMKII-p) expression was significantly upregulated, and Nav1.5, pThr17-phospholamban (PLB-t), pSer16-phospholamban (PLB-s), and ryanodine receptor (RYR2) expression was significantly downregulated in the ischemia–reperfusion (IR) zone in the non-therapeutic hypothermia (TH) group but not in the TH group. Because of technical issues and various protein sizes, Western blots protein analyses were conducted at different gels. The illustrative examples of Cx43 and NP-Cx43 were acquired from no-TH heart 1 and TH heart 1; the examples of CaMKII, CaMKII-P, PLB, PLB-t, PLB-s were from no-TH heart 2 and TH heart 2; the examples of Nav1.5 were from no-TH heart 3 and TH heart 3; the examples of sarcoplasmic reticulum Ca-ATPase (SERCA2a) were from no-TH heart 4 and TH heart 4; the examples of ryanodine receptor (RYR2) were from no-TH heart 5 and TH heart 5.

Fig 3: Activated CD47 increases CaMKII expression to increase HDAC3. A, Western immunoblot analysis of CaMKII and phosphorylated CaMKII (p-CaMKII) protein expression in LV tissue samples from patients with nonischemic end-stage LVHF (n=4) and normal controls (n=5). Densitometry is presented as the mean (±SEM). *=statistically significant difference (P<0.05), compared to control. B, Western immunoblot analysis of CaMKII and p-CaMKII protein expression in left ventricular tissue samples from wild-type (WT) and CD47-null animals 4 weeks post-TAC or controls. Densitometry is presented as the mean (±SEM) of n=6 to 9 animals/group. *=statistically significant difference (P<0.05), compared to control. C, Rat neonatal cardiac myocytes (RNCMs) were treated with peptide 7N3 (10 µmol/L) for the indicated time points, and Western immunoblot analysis of p-CaMKII and total CaMKII protein expression was performed. Densitometry is presented as the mean (±SEM) of 3 separate experiments. *=statistically significant difference (P<0.05), compared to control. RNCMs were treated with either a CD47 antibody (D; OX101, 1 mg/mL for 48 hours) or the specific CaKMII inhibitor, AIP (E; 10 nmol/L, 30 minutes)±peptide 7N3 (10 µmol/L, 3 hours), and Western immunoblot analysis of p-CaMKII, HDAC3, and phosphorylated HDCA3 (p-HDAC3) was performed. Densitometry is presented as the mean (±SEM) of 3 separate experiments. *=statistically significant difference (P<0.05), compared to 7N3+CD47 Ab treated and 7N3+AIP. F, RNCMs were loaded with the calcium indicator, Fluo-4 AM, treated with peptide 7N3 (10 µmol/L)±tetracaine (0.5 mmol/L) for the indicated time points, and images were acquired. Fluorescent intensity was determined and results are expressed as the mean±(SEM) from the analysis of cells within a minimum of 7 to 11 regions of interest per culture dish per treatment group. Significant difference (P<0.05) between treatment groups 7N3, compared to 7N3+tetracaine, from 17 through 30 minutes. G, Western immunoblot analysis of total and phosphorylated PLB (p-PLB) protein expression in left ventricular tissue samples from WT and CD47-null animals 4 weeks post-TAC or controls. Densitometry is presented as the mean (±SEM) of n=4 to 6 animals/group. *=statistically significant difference (P<0.05), compared to control; #=statistically significant difference (P<0.05), compared to WT TAC. AIP indicates autocamtide-2-related inhibitory peptide; CaMKII, calcium/calmodulin-dependent protein kinase II; LVHF, left ventricular heart failure; TAC, transverse aortic constriction.

Fig 4: Global transcriptome profile of Atxn2-CAG100-KIN mouse cerebellum at pre-onset stage. (A) Volcano plot analysis of all the quantified mRNAs showing significant up- or downregulations beyond 20% fold-change in red and green, respectively. Highly significant and cerebellar pathology-relevant transcripts among downregulations are colored in magenta. Transcriptome analysis was performed with Affymetrix Clariom D oligonucleotide microarray technology, comparing 3 Atxn2-CAG100-KIN cerebellum samples with 3 age- and sex-matched WT controls. Statistical assessment of the expression data was done using the Affymetrix Transcriptome Analysis Console; (B) Representative diagrams depicting the numbers of all mRNA transcripts measured (20,299), versus those that are dysregulated more than 35%, regardless of statistical significance (253 down, 147 up), with the number of significant alterations among all (2162), and the numbers of nominally significant (p < 0.05) dysregulations in either direction more than 35% (209 down, 94 up); (C) Protein interaction network of the significantly downregulated transcripts (<-1.35 fold) generated with STRING database (https://string-db.org/) revealed a core network with two central nodes of Itpr1 and Camk2a. Many factors in the network contributed to significantly altered pathways and cellular processes identified by Functional Enrichment Analysis function of STRING, and are highlighted in different colors corresponding to the colored bars in the subsequent panel D; (D) Functional Enrichment Analysis results of STRING utilizing GO terms Biological Process, KEGG Pathways, Reactome Pathways, UniProt Keywords and GO terms Molecular Function. Top ten dysregulated pathways and cellular processes are depicted per database for clarity, and complete lists are available in Supplementary Table S2. Colored bars represent pathways corresponding to similarly colored proteins in panel C.

Fig 5: Expression analyses of Ca2+ associated subcellular signaling pathways in Atxn2-CAG100-KIN cerebellum throughout disease course. (A) Transcript levels of cytoplasmic IP3 metabolism components (Gria3, Grm1, Grm4, Plcb3, Plcb4, Plcg1, Inpp5a, Itpka, Prkcd), Ca2+-CaM signaling components (Pcp4, Camkk1, Camkk2, Camk2a, Camk2b, Camk2d, Camk2g, Camk4) and downstream CaMKIV targets (Khdrbs1-3) were quantified by RT-qPCR in Atxn2-KO (blue bars) and Atxn2-CAG100-KIN mouse cerebellum at pre-onset (pink bars) and terminal (red bars) disease stages; (B) Protein levels of GluA3, CaMKIIa, CaMKIV and Sam68 in soluble (RIPA) and insoluble (SDS) fractions of Atxn2-CAG100-KIN mouse cerebellum at pre-onset and final disease stages were determined by quantitative immunoblots. Student’s t-test with Welch’s correction; 0.05 < p < 0.1 T, p < 0.05 *, p < 0.01 **, p < 0.001 ***, p < 0.0001 ****. Further information regarding individual fold changes and p-values can be found in Supplementary Table S3.