Additional file 1 of Diastolic dysfunction in a pre-clinical model of diabetes is associated with changes in the cardiac non-myocyte cellular composition
Creators: name: Cohen, Charles D.
name: De Blasio, Miles J.
name: Lee, Man K. S.
name: Farrugia, Gabriella E.
name: Prakoso, Darnel
name: Krstevski, Crisdion
name: Deo, Minh
name: Donner, Daniel G.
name: Kiriazis, Helen
name: Flynn, Michelle C.
name: Gaynor, Taylah L.
name: Murphy, Andrew J.
name: Drummond, Grant R.
name: Pinto, Alexander R.
name: Ritchie, Rebecca H.
Publisher: name: figshare
Year: 2021
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Physiology
subject: 59999 Environmental Sciences not elsewhere classified
subject: FOS: Earth and related environmental sciences
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: 69999 Biological Sciences not elsewhere classified
subject: 80699 Information Systems not elsewhere classified
subject: FOS: Computer and information sciences
subject: 60506 Virology

description: Additional file 1: Table S1. Physiological endpoint characteristics of STZ-HFD-induced murine diabetes. Table S2. Endpoint M-Mode echocardiography for assessing cardiac systolic function in murine diabetes. Table S3. Organ weights. Table S4. Flow cytometry antibody panel utilised in whole blood from mice. Table S5. Flow cytometry antibody panel utilised in myocardium from mice. Figure S1. (A) Representative images of transmitral annular blood flow via Doppler echocardiography. Quantified Doppler flow; (B) Anaesthetised heart rate (HR), (C) peak E-wave velocity, (D) A-wave velocity (E) E:A ratio, (F) deceleration time (DT) and (G) isovolumic relaxation time (IVRT). (H) Shows representative images for tissue Doppler echocardiography, quantified in figures I-L. (I) Peak e' velocity (P=0.054), (J) peak a' velocity (P = 0.072), (K) e':a' ratio, (L) E:e' ratio. ND = non-diabetic, DM = diabetes mellitus. Data presented as mean ± SEM and individual data points, and analysed using an unpaired t-test. Statistical significance was assumed at P &lt;0.05. Figure S2. Flow cytometry gating strategies – Heart. A) Illustrates the gating strategies used for identification of cardiac non-myocyte cell populations. Single, intact cells are first identified by the FSC-A/FSC-H gate. Next, cells are deemed ‘live and metabolically active’ by gating all SYTOX-Calcein+ events as indicated. Live, metabolically active cells were then identified based on their cell clustering to each respective antibody (listed on the x and y-axes). ECs = Endothelial cells, RMCs = Resident mesenchymal cells, Leuks = Leukocytes, Undefined = Undefined cells, Macs = Macrophages, MHCIIhi/lo = MHCIIhi/lo macrophages, Fibros = Fibroblasts, Mural = Mural cells, SMCs = Smooth Muscle Cells, Schwann = Schwann cells, FSC-A = forward scatter area, FSC-W = forward scatter width, FSC-H = forward scatter height. B) Depicts the total number of live, metabolically active cells acquired per sample (from flow cytometry), split by treatment (ND = non-diabetic, DM = diabetes mellitus. Data is presented as individual values. Each line indicates the median. P = NS (Student’s unpaired t-test). Figure S3. Flow cytometry gating strategies – Blood. Illustrates the gating strategies used for identification of circulating leukocyte populations. Single, intact cells are first identified by the FSC-A/FSC-H gate. Live cells were identified as DAPI- (4′,6-diamidino-2-phenylindole), after which cells are assigned as described in Supplementary Figure 2. FSC-A = forward scatter area, FSC-W = forward scatter width, FSC-H = forward scatter height. Figure S4. Flow cytometry gating strategies – Bone marrow. Illustrates the gating strategies used for identification of bone marrow progenitors. Lin- = lineage negative, HSPC = haematopoietic stem cell, LSK = lineage- cKit+ cells, FSC-H = forward scatter height. Lineage cocktail = CD3, CD19, CD2, B220, TER119, CD11b, Gr-1, CD8, CD4. Figure S5. Flow cytometry gating strategies – Spleen. Illustrates the gating strategies used for identification of splenic monocytes. SSC-A = side scatter area. Figure S6. Histological identification of resident mesenchymal cells. Representative micrograph of murine left-ventricle stained with PCM1 and GATA4 antibodies, counter-stained with DAPI. Monochrome images (left) indicate the positive signals acquired for nuclei enumeration. Each channel is then merged and displayed in colour (right). Figure S7. Histological identification of endothelial cells. Representative micrograph of murine left-ventricle stained with DACH1 and counter-stained with DAPI. Monochrome images (left) indicate the positive signals acquired for nuclei enumeration. Both channels are then merged and displayed in colour (right). Figure S8. Chronic hyperglycaemia is evident in diabetic mice throughout study duration. Hyperglycaemia was first detected 2-weeks after the commencement of STZ-HFD administration, and remains elevated until endpoint (measured fortnightly). Data is presented as mean ± SEM. Statistical significance was determined by a repeated measures ANOVA using a Tukey’s multiple comparison post-hoc test. **P &lt; 0.01, ***P &lt; 0.001, ****P &lt; 0.0001.