SRC-2 coactivator deficiency decreases functional reserve in response to pressure overload of mouse heart

Abstract

A major component of the cardiac stress response is the simultaneous activation of several gene regulatory networks. Interestingly, the transcriptional regulator steroid receptor coactivator-2, SRC-2 is often decreased during cardiac failure in humans. We postulated that SRC-2 suppression plays a mechanistic role in the stress response and that SRC-2 activity is an important regulator of the adult heart gene expression profile. Genome-wide microarray analysis, confirmed with targeted gene expression analyses revealed that genetic ablation of SRC-2 activates the "fetal gene program" in adult mice as manifested by shifts in expression of a) metabolic and b) sarcomeric genes, as well as associated modulating transcription factors. While these gene expression changes were not accompanied by changes in left ventricular weight or cardiac function, imposition of transverse aortic constriction (TAC) predisposed SRC-2 knockout (KO) mice to stress-induced cardiac dysfunction. In addition, SRC-2 KO mice lacked the normal ventricular hypertrophic response as indicated through heart weight, left ventricular wall thickness, and blunted molecular signaling known to activate hypertrophy. Our results indicate that SRC-2 is involved in maintenance of the steady-state adult heart transcriptional profile, with its ablation inducing transcriptional changes that mimic a stressed heart. These results further suggest that SRC-2 deletion interferes with the timing and integration needed to respond efficiently to stress through disruption of metabolic and sarcomeric gene expression and hypertrophic signaling, the three key stress responsive pathways.

Figure 1. Loss of SRC-2 results in extensive gene expression changes in the heart.

A, Heatmap and list of genes significantly up and downregulated in WT and SRC-2 KO hearts at FDR<0.05. Each column represents RNA isolated from an individual mouse (WT n = 4, KO n = 4). Rows correspond to gene IDs as listed in Supplemental Table 1. B, Immunoblot for SRC-2 in heart tissue lysates made from mouse hearts harvested at the indicated ages of development. GAPDH is used as a loading control.

Figure 2. Ablation of SRC-2 in the mouse mimics the metabolic gene expression of a stressed heart.

A–C, Quantitative PCR analysis (qPCR) of gene expression of the indicated gene involved in glycolytic (A), fatty acid (B), and Krebs cycle and oxidative phosphorylation (C), metabolic pathways. RNA was isolated from WT and SRC-2 KO hearts (n = 5). Individual gene expression is analyzed by ΔΔCt method with 18S RNA expression used as a normalizer and expression relative to WT. D, Lactate levels in WT and SRC-2 KO mouse heart tissue lysates. * = p≤0.05, ** = p≤0.01, and *** = p≤0.001. (Slc2a1- facilitated glucose transporter member 1 (GLUT1)^#, Slc2a4- facilitated glucose transporter member 4 (GLUT4)^#, Ldhd- lactate hydrogenase isoform d^#, Pfkfb1- 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 1^#, Fbp2- fructose 1,6-bisphophatase 2^#, Pkm2- pyruvate kinase, muscle, Pdk2 and 4- pyruvate dehydrogenase kinase 2^# and 4^#, Fabp5, 7, and 1- fatty acid binding proteins 5^#, 7^#, and 1^#, CD36- fatty acid transporter, AcsL1- acyl-CoA synthetase long-chain 1; Dgat 1 and 2- diacylglycerol O-acyltransferase 1 and 2, Acadl- long chain acyl-Coenzyme A dehydrogenase, Idh2- isocitrate dehydrogenase 2, Fh1- fumarate hydratase 1, ATPaf2- ATP synthase mitochondrial F1 complex assembly factor 2, COX4- cytochrome c oxidase subunit IV isoform 1, ATP5b- ATP synthase F1β subunit). A # indicates a gene identified in the microarray.

Figure 3. Shifts in sarcomeric and stress response gene expression profiles of SRC-2 KO.

A–B, qPCR analysis of the indicated actin, myosin, and tubulin isoforms (A), and cardiac stress response (B) genes. RNA was isolated from WT and SRC-2 KO hearts (n = 5). Individual gene expression is analyzed by ΔΔCt method with 18S RNA expression used as a normalizer and expression relative to WT. * = p≤0.05 and ** = p≤0.01. (Actc1- cardiac actin, Acta2- smooth muscle actin^#, Myh-myosin heavy chain 6 and 7, Mlc- myosin light chain (Myl2v), Tppp3- tubulin polymerization promoting protein family member 3^#, Tuba8- tubulin, alpha 8^#, c-myc- myelocytometosis oncogene, ANF- atrial natiuretic factor, Serca 2- cardiac muscle Ca+2 transporting ATPase). A # indicates a gene identified in the microarray.

Figure 4. Loss of SRC-2 results in decreased expression of several cardiac transcription factors important for controlling metabolic and sarcomeric gene expression.

A–D and F, qPCR analysis of the indicated transcription factor and transcription co-activator genes. RNA was isolated from WT and SRC-2 KO hearts (n = 5). Individual gene expression is analyzed by ΔΔCt method with 18S RNA expression used as a normalizer and expression relative to WT.E, Immunoblot for GATA-4 and MEF2 protein expression in WT and SRC-2 KO heart tissue lysates (n = 3).* = p≤0.05, ** = p≤0.01, and *** = p≤0.001. (PPAR- peroxisome proliferator activated-receptor α, β/δ, and γ, PGC-1- peroxisome proliferator activated-receptor γ coactivator-1 α and β, SRC- steroid receptor coactivator 1 and 3, SRF- serum response factor, GATA- GATA binding protein 4 and 6, MEF2c- myocyte enhancer factor 2c, Hand 2- heart and neural crest derivatives 2, Nkx2.5- NK2 transcription factor related 5, Gfat- glutamine frustose-6-phosphate transaminase 1 and 2, Tbx5- T-box 5^#, NRF1- nuclear respiratory factor 1, ERR- estrogen-related receptor α). A # indicates a gene identified in the microarray.

Figure 5. SRC-2 KO mice have no impairment in cardiac function in unstressed conditions.

A, Total heart weight normalized to tibia length for WT and SRC-2 KO mice (n = 5). B, Diastolic left ventricle wall thickness measured by Echocardiography in WT and SRC-2 KO mice (WT n = 15, KO n = 12). C, Cardiac Doppler measurements of blood flow rates (peak velocity, mean acceleration, and fractional shortening) and diastolic left ventricle interior diameter measured by Echocardiography in WT and SRC-2 KO mice (WT n = 15, KO n = 12).

Figure 6. TAC in SRC-2 KO mice results in both greater decline of cardiac function and failure to hypertrophy.

A, qPCR analysis of SRC-2 in WT sham and banded animals after 6 weeks of TAC (n = 4). Individual gene expression is shown normalized to 18S RNA expression. B, Immunoblot analysis of SRC-2 protein levels in sham and banded animals after 6 weeks TAC. GAPDH is used as a loading control. Numbers under the blot indicate densitometry measurements of SRC-2 versus GAPDH expression for each mouse. * = p≤0.05. C, Ratio of cardiac Doppler measurements of flow rates for right and left carotid to analyze amount of restriction due to TAC in WT and SRC-2 KO mouse groups (WT n = 16, KO n = 14). D, Cardiac Doppler measurements of blood flow rates (peak velocity and mean acceleration) in WT and SRC-2 KO hearts before and after 6 weeks TAC (WT n = 11, KO n = 7). Percent change as a result of banding is shown for each group. All Echo and Doppler “pre-” measurements are the same as presented in Figure 5. E, qPCR analysis of sham and TAC WT and SRC-2 KO hearts 6 weeks post-TAC for the indicated genes (n = 4). Individual gene expression is analyzed by ΔΔCt method with 18S RNA expression used as a normalizer and expression relative to WT. * = p≤0.05, ** = p≤0.01 and *** = p≤0.001.

Figure 7. Hypertrophic signaling pathways are impaired in SRC-2 KO hearts.

A, Echocardiography analysis of diastolic left ventricle wall thickness before and after 6 weeks TAC in WT and SRC-2 KO mice (WT n = 11, KO n = 7). Percent change as a result of banding is shown for each group. Measurement of WT and SRC-2 KO total heart weight normalized to tibia length in unstressed animals and post- 6 weeks TAC (WT n = 11, KO n = 7). All Echo and Doppler “pre-” measurements are the same as presented in Figure 5. B-C, Immunoblot analysis of the indicated proteins in WT and SRC-2 KO heart tissue lysates made on hearts 6 weeks post-TAC. GAPDH is used as a loading control. Each graph represents quantitation of the blot for each lane compared to its own GAPDH and then normalized to one WT sample (n = 3). * = p≤0.05 and ** = p≤0.01.