Pyruvate kinase splice variants in fibroblasts influence cardiac remodeling after myocardial infarction in male mice

Wells, Collin K.1; Nguyen, Daniel C.1; Brainard, Robert E.1; McNally, Lindsey A.1; De Silva, Maleesha1; Brittian, Kenneth R.1; Garrett, Lauren1; Taylor, Madison S.1; Martinez-Ondaro, Yania1; Howard, Caitlin1; Suluru, Snigdha1; Dassanayaka, Sujith1; Mohamed, Tamer M.A.1 2; Singhal, Richa1; Gibb, Andrew A.1; Lorkiewicz, Pawel K.1; Moore, Joseph B.1; Jones, Steven P.1; Hill, Bradford G.1

Published Mar 09, 2026 on Dryad. https://doi.org/10.5061/dryad.tx95x6b8w

Abstract

Fibroblasts are crucial for cardiac repair after myocardial infarction (MI). In response to signaling cues, they differentiate to phenotypes with robust capacities to synthesize and secrete extracellular matrix (ECM) and signaling molecules. Although activated fibroblast phenotypes are associated with pronounced changes in metabolism, it remains unclear how the metabolic network upholds the effector functions of fibroblasts in the infarcted heart. We found that two enzymes that could facilitate a phosphoenolpyruvate cycle, i.e. pyruvate kinase muscle isoform 2 (PKM2) and phosphoenolpyruvate carboxykinase 2 (PCK2), are elevated in the heart after MI. Although Pck2 deletion had no effect on post-MI remodeling, fibroblast-specific switching of Pkm2 to Pkm1 (fbPkm2→1) mitigated ventricular dilation, wall thinning, and losses in ejection fraction caused by MI. Despite these salutary effects, fbPkm2→1 switching did not alter cardiac fibrosis in vivo, nor did it affect collagen production, cytokine or chemokine secretion, myofibroblast differentiation markers, or transcriptional regulation in vitro. Nevertheless, Pkm2→1 splice variant switching increased myofibroblast contractile activity as well as influenced the metabolic phenotype of fibroblasts, as shown by increased pyruvate kinase activity, higher mitochondrial respiratory capacity, and elevation in glycolytic intermediate abundance. Despite these changes, Pkm2→1 switching had relatively minor effects on glucose carbon fate, as determined by stable isotope-resolved metabolomics. Nevertheless, these metabolic data demonstrate that cardiac fibroblasts exhibit minimal glucose-supported de novo glycine synthesis in vitro, yet possess high hexosamine and glucuronate biosynthetic pathway activity. Collectively, these findings reveal that fibroblast PKM isoforms influence post-MI remodeling, highlighting pyruvate kinase as a potential therapeutic target. This dataset includes a spreadsheet organized by figure number that provides the raw data used for analysis in this study.

Dataset DOI: 10.5061/dryad.tx95x6b8w

Description of the data and file structure

The data included in this submission involve multiple data types, including data related to metabolism, gene expression, protein abundance and secretion, and cardiac function. The uploaded data include the raw data used to generate the graphs, with as little redundancy with the published manuscript as possible. In addition, RNA seq data were generated, which are available NCBI GEO database under Accession No. GSE291984.

Files and variables

File: Figure_1.csv

Description: PKM2 abundance increases in the infarcted heart and is found in areas of replacement fibrosis.

Immunoblots and relative quantification of PKM1 and PKM2 in whole heart homogenates 5 d after sham or MI surgery. n = 8 male mice per group. Proteins were normalized to amido black signal from stained membranes, with fold change relative to sham controls. Data are shown as means ± SEM, Student’s unpaired t-test.

Figure. 1A: Data are densitometry values obtained from westerns probing for PKM1 or PKM2. Imaged and analyzed on Image Lab (Bio-Rad Software)

Abbreviations:

Pkm1: Pyruvate kinase-muscle 1

Pkm2: Pyruvate kinase-muscle 2

MI: myocardial infarction

Variables:

Sham PKM1, Sham PKM2: Sham control mice, whole heart protein expression of PKM1 or PKM2

MI PKM1, MI PKM2: Mice received MI, whole heart protein expression of PKM1 or PKM2

File: Figure_2.csv

Description: Fibroblast-specific Pkm2➔1 splice variant switching improves cardiac remodeling and alleviates congestive heart failure after myocardial infarction.

Pkm2fl/fl mice were crossed with mice expressing tamoxifen (tamox)-inducible, fibroblast-specific Cre recombinase (Col1a2-CreERT) to render a fibroblast-specific Pkm2➔1 splice variant switch (fbPkm2➔1) upon tamoxifen administration (i.e. 20 mg/kg/d for 5 d, i.p.). n = 9–13 male mice per group. 28 days following myocardial infarction, transthoracic echocardiography endpoints (Figure 2D-F) of the left ventricle was measured using Vevo ultrasound platforms (FUJIFILM VisualSonics) and ratio of weighed wet to dry lung (Figure 2G) was obtained. For statistical analysis, unpaired student’s t-tests were performed. Data are shown as means ± SEM.

Figure. 2D: Values represent end diastolic volume (µL)

Figure. 2E: Values represent end systolic volume (µL)

Figure. 2F: Values represent ejection fraction (%)

Figure. 2G: Values represent wet/dry lung weight presented as a ratio

Variables:

Pkm2fl/fl: pyruvate kinase muscle isoform 2 non-recombined control mice

Pkm2->1: recombined Pkm2fl/fl mice

n/a: missing N, Pkm2fl/fl N = 12-13; fbPkm2->1 N = 9

File: Figure_3.csv

Description: Fibroblast-specific Pkm2➔1 splice variant switching does not affect collagen abundance or macrostructural attributes.

Each heart was dissected into blocks (B1–B4) prior to histopathological staining and analysis. Planimetric measurements of picrosirius red-stained myocardial tissue sections were used to quantify fibrotic content and scar width values. Scars width values from sections B3 & B4 were averaged to generate one value for each biological replicate. Hearts were sectioned into 1-mm cross-sectional slices and fixed in 10% formalin. Picrosirius Red (Direct Red 80, Sigma-Aldrich #365548) was used to detect collagen. Sections were visualized using a Keyence Imaging System. n = 5–9 male mice per group. For statistical analysis, Student’s t-tests were performed. Data are shown as means ± SEM. Second harmonic generation (SHG) imaging was performed on B4 (Apex) myocardial tissue sections to interrogate collagen macrostructural attributes within the infarct region. n = 5–9 male mice per group. For statistical analysis, Student’s t-tests were performed. Data are shown as means ± SEM.

Figure 3C: Values represent fibrotic content (%) of each tissue section (B1-B4)

Figure 3D: Values represent the average scar width (µm) of tissue sections B3 and B4

Figure 3F: Values represent collagen fiber alignment

Figure 3G: Values represent collagen fiber straightness

Figure 3H: Values represent collagen fiber width (µm)

Abbreviations:

B (1, 2, 3, 4): Block 1, Block 2, Block 3, Block 4. Block 1 being myocardial tissue section closest to base of heart, whereas Block 4 is the section closest to the apex.

Variables

Pkm2fl/fl: pyruvate kinase muscle isoform 2 non-recombined control mice

Pkm2->1: recombined Pkm2fl/fl mice

B1: Myocardial tissue section, block 1

B2: Myocardial tissue section, block 2

B3: Myocardial tissue section, block 3

B4: Myocardial tissue section, block 4

n/a: missing N, Pkm2fl/fl N = 5; fbPkm2->1 N = 9

File: Figure_4.csv

Description: Pkm2➔1 splice variant switching does not affect myofibroblast or inflammatory fibroblast differentiation but does impact contractile function

Cardiac fibroblasts isolated from Pkm2fl/fl mice were transduced with Ad-CMV-iCre or vector control (Ad-CMV-LacZ) for 48 h followed by treatment with bFGF (20 ng/ml), TGFβ (10 ng/ml), or IL-1β (15 ng/ml) for 48 h. Quantification of immunoblots of intracellular PKM splice variants and markers of myofibroblast or inflammatory fibroblast differentiation. Proteins were normalized to amido black signal from stained membranes. n = 4 biological replicates (male) per group. For statistical analysis, two-way ANOVA with Sidak’s post-hoc test was performed. Data are shown as means ± SEM. Representative group quantification of collagen gel contraction assay. Gel surface area was assessed 5 d post-ligand treatment. n = 4 biological replicates (male) per group. For statistical analysis, two-way ANOVA with Sidak’s post-hoc test was performed. Data are shown as means ± SEM.

Figure. 4B: Data are densitometry values obtained from westerns probing for PKM1, PKM2, PSTN, Col1a1, COX2, αSMA, and MMP3. Imaged and analyzed on Image Lab (Bio-Rad Software)

Figure. 4C: Data are quantification of surface area (arbitrary units) of gel in dish, analyzed through ImageJ.

Abbreviations:

Pkm1: pyruvate kinase-muscle 1

Pkm2: pyruvate kinase-muscle 2

PSTN: periostin

Col1a1: collagen type I alpha 1 chain

COX2: cyclooxygenase-2

aSMA: alpha-smooth muscle actin

MMP3: matrix metallopeptidase 3

SS: Serum Starved

bFGF: basic fibroblast growth factor

TGFB: transforming growth factor beta

IL-1B: interleukin-1beta

LacZ: Ad-CMV-Lacz (adenovirus vector control)

iCre: Ad-CMV-iCre (improved Cre adenovirus)

Variables:

LacZ: Fibroblasts treated with adenovirus vector control

iCre: Fibroblasts treated with improved Cre adenovirus

SS: Fibroblasts treated with serum starved media (vehicle control)

bFGF: Fibroblasts treated with basic fibroblast growth factor

TGFB: Fibroblasts treated with transforming growth factor-beta

IL1-B: Fibroblasts treated with interleukin-1beta

File: Figure_5.csv

Description: Effect of Pkm2➔1 isoform switching on pyruvate kinase activity, glycolysis, and mitochondrial metabolism in cardiac fibroblasts.

Adult cardiac fibroblasts were isolated from Pkm2fl/fl mice and treated with adenoviruses encoding iCre or control vector (LacZ). Pyruvate kinase (PK) activity: After 48 h, the cells were treated with either bFGF (20 ng/ml) or TGFβ (10 ng/ml) for 48 h and pyruvate kinase activity assay was assessed (n = 4 male mice per group). For statistical analysis, two-way ANOVA with Sidak’s post-hoc test was performed. n = 4 biological replicates (male) per group. Data are shown as means ± SEM. Extracellular flux analysis: Fibroblasts were treated were treated with bFGF (20 ng/ml) or TGFβ (10 ng/ml) for 48 h and then subjected to extracellular flux (XF) analysis. For XF analysis, cells were subjected to a mitochondrial stress test and treated with the following compounds: oligomycin (Oligo), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), antimycin A and rotenone (AA/Rot). Oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and bioenergetic parameters were quantified. n = 3 biological replicates (male) per group. Data are shown as means ± SEM.

Figure 5A: Data are Pyruvate Kinase enzymatic activity, represented as nmol/min/ug, acquired through kit from Abcam (ab843432)

Figure 5B: Data are extracellular flux traces analyzed via Seahorse XF96 (Agilent). The oxygen consumption rate (OCR; pmol/min/µg protein) and extracellular acidification rate (ECAR; mpH/min/ µg protein) values were normalized to total protein measurements (via Lowry assay) in each well.

Figure 5C: Data are bioenergetic parameters (basal OCR, proton leak, ATP-linked respiration, max OCR, spare respiratory capacity, non-mitochondrial-linked respiration, basal ECAR, and oligomycin-sensitive ECAR) calculated from OCR or ECAR traces.

Abbreviations:

OCR: oxygen consumption rate

ECAR: extracellular acidification rate:

Proton: proton leak

ATP: ATP-linked respiration

Max: Max OCR

Spare: spare respiratory capacity

Non-Mito: non-mitochondrial-linked respiration

Oligo-Sens.: oligomycin-sensitive ECAR

Variables:

LacZ/bGal FGF: Fibroblasts treated with adenovirus vector control and basic fibroblast growth factor

LacZ/bGal TGFB: Fibroblasts treated with adenovirus vector control and transforming growth factor beta

iCre FGF: Fibroblasts treated with improved Cre adenovirus and basic fibroblast growth factor

iCre TGFB: Fibroblasts treated with improved Cre adenovirus and transforming growth factor beta

File: Figure_6.csv

Description: Pkm2➔1 splice variant switching increases the abundance of 3 carbon glycolytic intermediates in myofibroblasts.

Cardiac fibroblasts were isolated from Pkm2fl/fl mice and treated with adenoviruses encoding iCre or LacZ (control). After 48 h, the fibroblasts were treated with bFGF (20 ng/ml) or TGFβ (10 ng/ml) for 48 h. Cells were then cultured with [U-13C]-glucose for 12 h. Polar fractions were subjected to liquid chromatography/mass spectrometry analysis. 13C enrichment values were transformed via logit transformation prior to statistical analysis. Relative pool abundance values were normalized to total protein prior to statistical tests. n = 4 male mice per group. For statistical analyses, two-way ANOVA with Sidak’s post-hoc test was performed. Data are shown as means ± SEM.

Figure 6B: Data are Total 13C fractional enrichment and relative pool abundance of glycolytic intermediate metabolites.

Abbreviations:

Hexose-1,6-BP: hexose-1,6-bisphosphate

DHAP: dihydroxyacetone phosphate

3-PG: 3-phosphoglycerate

PEP: phosphoenolpyruvate

Variables:

LacZ bFGF: Fibroblasts treated with adenovirus vector control and basic fibroblast growth factor

LacZ TGFB: Fibroblasts treated with adenovirus vector control and transforming growth factor beta

iCre bFGF: Fibroblasts treated with improved Cre adenovirus and basic fibroblast growth factor

iCre TGFB: Fibroblasts treated with improved Cre adenovirus and transforming growth factor beta

Fractional Enrichment: Total 13C fractional enrichment of given metabolite

Pool Abundance: Relative pool abundance of given metabolite

Null: represents values not detected/below threshold

File: Figure_7.csv

Description: Effect of Pkm2➔1 splice variant switching on accessory pathways of glucose metabolism.

Figure 7B: Data are Total 13C fractional enrichment and relative pool abundance of metabolites in accessory pathways of glucose metabolism.

Abbreviations:

Ribose-5-P: ribose 5-phosphate

Sedoheptulose-7-P: sedoheptulose-7-phosphate

UDP-HexA: UDP-hexuronate

UDP-HexNAc: UDP-N-acetylhexosamine.

Variables:

LacZ bFGF: Fibroblasts treated with adenovirus vector control and basic fibroblast growth factor

LacZ TGFB: Fibroblasts treated with adenovirus vector control and transforming growth factor beta

iCre bFGF: Fibroblasts treated with improved Cre adenovirus and basic fibroblast growth factor

iCre TGFB: Fibroblasts treated with improved Cre adenovirus and transforming growth factor beta

Fractional Enrichment: Total 13C fractional enrichment of given metabolite

Pool Abundance: Relative pool abundance of given metabolite

Null: represents values not detected/below threshold

File: Figure_8.csv

Description: Pkm2➔1 splice variant switching does not affect transcription in cardiac fibroblasts

Cardiac fibroblasts were isolated from Pkm2fl/fl mice and treated with adenoviruses encoding iCre or LacZ control. After 48 h, the fibroblasts were treated with bFGF (20 ng/ml), TGFβ (10 ng/ml), or IL-1β (15 ng/ml) for 24 h. RNA was then isolated and subjected to bulk RNA sequencing. n = 4 male mice per group.

Figure 8C: Data are quantified upregulated and downregulated DEGs; comparisons shown are bFGF:SS, IL1-β:SS, and TGFβ:SS for LacZ treated fibroblasts

Variables:

LacZ SS: Fibroblasts treated with adenovirus vector control and serum starved media (vehicle control)

LacZ FGF: Fibroblasts treated with adenovirus vector control and basic fibroblast growth factor

LacZ IL1B: Fibroblasts treated with adenovirus vector control and interleukin 1-beta

LacZ TGFB: Fibroblasts treated with adenovirus vector control and transforming growth factor beta

iCre bFGF: Fibroblasts treated with improved Cre adenovirus and basic fibroblast growth factor

iCre IL1B: Fibroblasts treated with improved Cre adenovirus and interleukin 1-beta

iCre TGFB: Fibroblasts treated with improved Cre adenovirus and transforming growth factor beta

iCre SS: Fibroblasts treated with improved Cre adenovirus and serum starved media (vehicle control)

ALL: Both up- and down-regulated genes

UP: up-regulated genes

DOWN: down-regulated genes

File: Suppl_2.csv

Description: Survival of fbPkm2➔1 and Pkm2fl/fl male and female mice post-infarct

Conditional mice bearing floxed Pkm2 alleles (Pkm2fl/fl) were bred to transgenic mice overexpressing Cre-recombinase fused to mutant estrogen receptor ligand-binding domains under the control of the collagen alpha-2(I) chain (Col1a2) promoter (Col1a2-CreER). To induce Cre recombination and subsequent deletion of Pkm2, adult mice (12 weeks of age) were injected with tamoxifen for five consecutive days. Adult,12–20-week-old, male and female mice were subjected to myocardial infarction (MI). MI was induced by permanent ligation of the left coronary artery. Statistics: log-rank (Mantel-Cox) test

Suppl. Fig. 2: Data are survival data of mice following MI. “1” = Mouse died on corresponding “Days post-MI”; “0” = Mouse survived the complete 28 d duration.

Variables:

Male Pkm2fl/fl, Female Pkm2fl/fl: Male/female pyruvate kinase muscle isoform 2 non-recombined control mice.

Male fbPkm2->1, Female fbPkm2->1: Male/female recombined Pkm2fl/fl mice.

Days post-MI: Days after myocardial infarction surgery that mice survived.

n/a: empty cell

File: Suppl_3.csv

Description: PCK2 is dispensable for cardiac remodeling following acute myocardial infarction.

Relative quantification of immunoblots of PCK2 in whole heart homogenates 5 d after sham or MI surgery. n=8 adult male mice per group. As well as key echocardiographic endpoints 28 d following permanent coronary ligation of wildtype and Pck2 knockout mice. n=9-16 adult male mice per group. For statistical considerations, Student’s T-tests were performed. Data are shown as means ± SEM

Suppl. Fig. 3C: Data are densitometry values obtained from westerns probing for PCK2. Imaged and analyzed on Image Lab (Bio-Rad Software)

Suppl. Fig. 3E: Values represent heart rate (BPM)

Suppl. Fig. 3F: Values represent end diastolic volume (µL)

Suppl. Fig. 3G: Values represent end systolic volume (µL)

Suppl. Fig. 3H: Values represent ejection fraction (%)

Abbreviations:

MI: myocardial infarction

Pck2: Phosphoenolpyruvate Carboxykinase-2

Variables:

SHAM: Mice that underwent sham surgery (control)

MI: Mice that underwent permanent coronary ligation, or MI (myocardial infarction)

Pck2+/+: Control wildtype mice possessing Pck2

Pck2-/-: Mice possessing homozygous knockout of Pck2

n/a: Represents missing N, Pck2+/+ N = 9; Pck2-/- N = 16

File: Suppl_4.csv

Description: Survival of Pck2+/+ and Pck2-/- male and female mice post-infarct.

The global Pck2 knockout mice were generated at the University of Texas Southwestern Mouse Genome Engineering Facility. Briefly, the global knockouts were a biproduct for the attempts to use CRISPR editing to flank exon 5 with two flox sites. Adult,12–20-week-old, male and female mice were subjected to myocardial infarction (MI). MI was induced by permanent ligation of the left coronary artery. Statistics: log-rank (Mantel-Cox) test

Suppl. Fig. 4: Data are survival data of mice following MI. “1” = Mouse died on corresponding “Days post-MI”; “0” = Mouse survived the complete 28 d duration.

Variables:

Male Pck2+/+, Female Pck2+/+: Male/female Phosphoenolpyruvate carboxykinase 2 wildtype mice (control).

Male Pck2-/-, Female Pck2-/-: Male/female homozygous Pck2 knockout mice.

Days post-MI: Days after myocardial infarction surgery that mice survived.

n/a: empty cell

File: Suppl_5.csv

Description: Pck2 deletion only modestly impacts cardiac fibroblast phenotype.

To assess proliferation, Prestoblue™ assay was performed using isolated cardiac fibroblasts from adult, male Pck2-/- and Pck2+/+ mice. Fluorescence intensity was measured 1 d following plating (t = 0 d) and once a day afterwards for 3 consecutive days. Isolated fibroblasts were also treated with transforming growth factor-β and basic fibroblast growth factor for 48 h prior to immunoblot detection and extracellular flux analysis.

Suppl. Fig. 5A: Data are measurements of fluorescence intensity (RFU) assessing proliferation.

Suppl. Fig. 5C: Data are densitometry values obtained from westerns probing for PCK2, αSMA, and Periostin. Imaged and analyzed on Image Lab (Bio-Rad Software)

Suppl. Fig. 5D: Data are extracellular flux traces analyzed via Seahorse XF96 (Agilent). The oxygen consumption rate (OCR; pmol/min/µg protein) and extracellular acidification rate (ECAR; mpH/min/ µg protein) values were normalized to total protein measurements (via Lowry assay) in each well.

Abbreviations:

WT: Wildtype

KO: Knockout

PCK2: Phosphoenolpyruvate Carboxykinase-2

αSMA: alpha-smooth muscle actin

PSTN: Periostin

bFGF: Basic fibroblast growth factor

TGFB: Transforming growth factor-β

OCR: oxygen consumption rate

ECAR: extracellular acidification rate

Variables:

Day: number of day where PrestoBlue assay was performed and fluorescence intensity was measured to assess proliferation

WT: Wildtype control fibroblasts

KO: Pck2 knockout fibroblasts

Pck2+/+: Wildtype control fibroblasts

Pck2+/+: Pck2 knockout fibroblasts

bFGF: Cells treated with bFGF

TGFB: Cells treated with TGFβ

File: Suppl_6.csv

Description: PKM1 and PKM2 are secreted proteins

Immunoblots of PKM1 and PKM2 present in culture media: Cardiac fibroblasts isolated from Pkm2fl/fl mice were transduced with Ad-CMV-iCre or vector control (Ad-CMV-LacZ) for 48 h followed by treatment with no ligand (serum starved; SS), bFGF, or TGFβ for 48 h. Media was collected and used for Immunoblots of extracellular PKM splice variants. n = 3 male mice per group. For statistical analysis, two-way ANOVA with Sidak’s post-hoc test was performed. Data are shown as means ± SEM.

Suppl. Fig. 6: Data are densitometry values obtained from westerns probing for extracellular PKM2 and PKM1. Imaged and analyzed on Image Lab (Bio-Rad Software)

Variables:

LacZ: Fibroblasts treated with adenovirus vector control

iCre: Fibroblasts treated with improved Cre adenovirus

Control: Fibroblasts treated with serum starved media (vehicle control)

bFGF: Fibroblasts treated with basic fibroblast growth factor

TGFB: Fibroblasts treated with transforming growth factor-beta

File: Suppl_7.csv

Description: Time course of 13C incorporation into glucose-derived metabolites in cardiac fibroblasts

Isolated cardiac fibroblasts were incubated with 13C6-glucose for 1, 6, 12, or 24 h followed by polar metabolite extraction and metabolite analysis via LC/MS.

Suppl. Fig. 7: Data are Total 13C fractional enrichment of various glucose-derived metabolites over a time course of 1, 6, 12, and 24 h.

Abbreviations:

DHAP: dihydroxyacetone phosphate

Sedoheptulose-7-P: sedoheptulose-7-phosphate

UDP-HexNAc: UDP-N-acetylhexosamine

ADP: Adenosine diphosphate

Variables:

Time: Amount of time (hours) labeled 13C-glucose tracer was incubated in cells

Mean: Mean fractional enrichment between replicates

SD: standard deviation

Replicates: number of replicates used

File: Suppl_8.csv

Description: Effect of Pkm2->1 splice variant switching on TCA cycle metabolites

Cardiac fibroblasts were isolated from Pkm2fl/fl mice and treated with adenoviruses encoding iCre or LacZ control. After 48 h, the fibroblasts were treated with bFGF or TGF-β for 48 h. Cells were cultured with [U-13C]-glucose for 12 h. Polar fractions were subjected to mass spectrometric analysis. 13C enrichment values were transformed via logit transformation prior to statistical analysis. Relative pool abundance values were normalized to total protein prior to statistical tests. n = 4 male mice per group. For statistical analyses, two-way ANOVA with Sidak’s post-hoc test was performed. Data are shown as means ± SEM.

Suppl. Fig. 8A: Data are Total 13C fractional enrichment and relative pool abundance of citrate

Suppl. Fig. 8B: Data are Total 13C fractional enrichment and relative pool abundance of isocitrate

Suppl. Fig. 8C: Data are Total 13C fractional enrichment and relative pool abundance of αKG

Suppl. Fig. 8D: Data are Total 13C fractional enrichment and relative pool abundance of succinate

Suppl. Fig. 8E: Data are Total 13C fractional enrichment and relative pool abundance of fumarate

Suppl. Fig. 8F: Data are Total 13C fractional enrichment and relative pool abundance of malate

Abbreviations:

aKG: α-ketoglutarate (αKG)

Variables:

LacZ bFGF: Fibroblasts treated with adenovirus vector control and basic fibroblast growth factor

LacZ TGFB: Fibroblasts treated with adenovirus vector control and transforming growth factor beta

iCre bFGF: Fibroblasts treated with improved Cre adenovirus and basic fibroblast growth factor

iCre TGFB: Fibroblasts treated with improved Cre adenovirus and transforming growth factor beta

Fractional Enrichment: Total 13C fractional enrichment of given metabolite

Pool Abundance: Relative pool abundance of given metabolite

File: Suppl_9.csv

Description: Pkm2->1 splice variant switching does not affect hyaluronan abundance.

Adult cardiac fibroblasts were isolated from Pkm2fl/fl mice and treated with adenoviruses encoding iCre or control vector (LacZ). After 48 h, the cells were treated with either bFGF (20 ng/ml) or TGFβ (10 ng/ml) for 48 h, and hyaluronan (HA) abundance was assessed via ELISA. Samples were diluted 1:3000 using provided dilution buffer to ensure they would fit within the standard curve. n = 4 biological replicates (male) per group. For statistical analysis, two-way ANOVA with Sidak’s posthoc test was performed. Data are shown as means ± SD.

Suppl. Fig. 9: Data are quantification of hyaluronan via ELISA assay (ng/ml)

Variables:

LacZ bFGF: Fibroblasts treated with adenovirus vector control and basic fibroblast growth factor

LacZ TGFB: Fibroblasts treated with adenovirus vector control and transforming growth factor beta

iCre bFGF: Fibroblasts treated with improved Cre adenovirus and basic fibroblast growth factor

iCre TGFB: Fibroblasts treated with improved Cre adenovirus and transforming growth factor beta

File: Suppl_10_11.csv

Description: Pkm2->1 splice variant switching does not affect cytokine, chemokine, or growth factor secretion.

Cardiac fibroblasts were isolated from Pkm2fl/fl mice and treated with adenoviruses encoding iCre or LacZ control. After 48 h, the fibroblasts were treated with bFGF or TGF-β for 48 h. Following treatment, the media was assayed with the 44-plex Mouse Cytokine Array/Chemokine Array. Data values that returned below or above the limit of detection (out of range) were omitted. n = 3-6 male mice per group. For statistical analysis, two-way ANOVA with Sidak’s post-hoc test was performed.

Suppl. Fig. 10-11: Data are quantification of extracellular cytokines (pg/mL/µg total protein)

Abbreviations:

G-CSF: Granulocyte Colony-Stimulating Factor

M-CSF: Macrophage Colony-Stimulating Factor

IFNB-1: Interferon Beta-1

IL-4: Interleukin-4

IL-5: Interleukin-5

IL-6: Interleukin-6

IL-11: Interleukin-11

IL-13: Interleukin-13

IL-16: Interleukin-16

IL-17: Interleukin-17

IL-20: Interleukin-20

IP-10: Interferon Gamma-Induced Protein 10

KC: Keratinocyte Chemoattractant

LIF: Leukemia Inhibitory Factor

LIX: Lipopolysaccharide-Induced CXC Chemokine

RANTES: Regulated upon Activation, Normal T Cell Expressed and Secreted

VEGF: Vascular Endothelial Growth Factor

EPO: Erythropoietin

MCP-1: Monocyte Chemoattractant Protein-1

MCP-5: Monocyte Chemoattractant Protein-5

MIP-1a: Macrophage Inflammatory Protein-1 alpha

MIP-3a: Macrophage Inflammatory Protein-3 alpha

Variables:

Replicate: Replicate identifier; replicate number