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Comparative Study
. 2012 Jan 3;125(1):65-75.
doi: 10.1161/CIRCULATIONAHA.111.050666. Epub 2011 Nov 15.

Glycogen synthase kinase-3α limits ischemic injury, cardiac rupture, post-myocardial infarction remodeling and death

Hind Lal  1 , Jibin Zhou, Firdos Ahmad, Raihana Zaka, Ronald J Vagnozzi, Morgan Decaul, James Woodgett, Erhe Gao, Thomas Force
Affiliations
Comparative Study

Glycogen synthase kinase-3α limits ischemic injury, cardiac rupture, post-myocardial infarction remodeling and death

Hind Lal et al. Circulation. .

Abstract

Background: The molecular pathways that regulate the extent of ischemic injury and post-myocardial infarction (MI) remodeling are not well understood. We recently demonstrated that glycogen synthase kinase-3α (GSK-3α) is critical to the heart's response to pressure overload. However, the role, if any, of GSK-3α in regulating ischemic injury and its consequences is not known.

Methods and results: MI was induced in wild-type (WT) versus GSK-3α((-/-)) (KO) littermates by left anterior descending coronary artery ligation. Pre-MI, WT, and KO hearts had comparable chamber dimensions and ventricular function, but as early as 1 week post-MI, KO mice had significantly more left ventricular dilatation and dysfunction than WT mice. KO mice also had increased mortality during the first 10 days post-MI (43% versus 22%; P=0.04), and postmortem examination confirmed cardiac rupture as the cause of most of the deaths. In the mice that survived the first 10 days, left ventricular dilatation and dysfunction remained worse in the KO mice throughout the study (8 weeks). Hypertrophy, fibrosis, and heart failure were all increased in the KO mice. Given the early deaths due to rupture and the significant reduction in left ventricular function evident as early as 1 week post-MI, we examined infarct size following a 48-hour coronary artery ligation and found it to be increased in the KO mice. This was accompanied by increased apoptosis in the border zone of the MI. This increased susceptibility to ischemic injury-induced apoptosis was also seen in cardiomyocytes isolated from the KO mice that were exposed to hypoxia. Finally, Bax translocation to the mitochondria and cytochrome C release into the cytosol were increased in the KO mice.

Conclusion: GSK-3α confers resistance to ischemic injury, at least in part, via limiting apoptosis. Loss of GSK-3α promotes ischemic injury, increases risk of cardiac rupture, accentuates post-MI remodeling and left ventricular dysfunction, and increases the progression to heart failure. These findings are in striking contrast to multiple previous reports in which deletion or inhibition of GSK-3β is protective.

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Figures

Figure 1
Figure 1
Deletion of GSK-3α increases ischemic injury and mortality post-MI. A, WT and GSK-3α KO mice were subjected to MI or sham surgery and survival was monitored for 8 weeks. Survival was analyzed by the Kaplan-Meier method, and differences between groups were determined by the Gehan-Breslow-Wilcoxon test. B, Two-month-old WT and KO mice were subjected to left coronary artery ligation or sham surgery. At 48 hours postligation, myocardial infarct size was determined by triphenyltetrazolium chloride staining. Infarct size was quantified and expressed as a percentage of the total area of the LV myocardium. Differences between groups were determined by unpaired t test, and results are expressed as means±SEM. A probability value of ≤0.05 was considered to denote statistical significance. MI indicates myocardial infarction; WT, wild type; KO, GSK-3α(−/−); LV, left ventricular.
Figure 2
Figure 2
Deletion of GSK-3α increases dilatative remodeling and cardiac dysfunction post-MI. Two-month-old WT and GSK-3α KO mice underwent baseline transthoracic echocardiographic examination. Twenty-four hours later they were subjected to occlusion of the proximal left anterior descending coronary artery. Mice were then followed with echocardiography at the time points shown. A, Left ventricular internal dimension at end-diastole (LVID;d). B, LVID at end-systole (LVID;s). C, left ventricular ejection fraction (LVEF). D, LV fractional shortening (LVFS). Repeated-measures analysis of variance was used to evaluate the statistical significance, and results are expressed as means±SEM. A probability value of ≤0.05 was considered to denote statistical significance. Probability values are for the comparison of WT versus KO mice subjected to MI. MI indicates myocardial infarction; WT, wild type; KO, GSK-3α(−/−).
Figure 3
Figure 3
Deletion of GSK-3α leads to cardiac and cardiomyocyte hypertrophy post-MI. Two-month-old WT and GSK-3α KO mice were subjected to MI or sham surgery for 3 weeks, as described in Materials and Methods. A, Increased hypertrophy in the KO mice subjected to coronary artery ligation as shown by HW/BW ratio. B, Increased HF in the KO mice. The ratio of lung weight to body weight (LW/BW, a measure of heart failure) was significantly increased in the KO mice. C, Representative images of LV remote myocardium 8 weeks post-MI stained with hematoxylin-eosin. D, Quantification of cardiomyocyte CSA. For determination of CSA, a minimum of 100 cardiomyocytes per heart were measured. Differences between groups were determined by unpaired t test, and results are expressed as means±SEM. A probability value of ≤0.05 was considered to denote statistical significance. MI indicates myocardial infarction; WT, wild type; KO, GSK-3α(−/−); CSA, cross-sectional area; HW, heart weight; LW, lung weight; BW, body weight.
Figure 4
Figure 4
Reactivation of fetal gene expression in the GSK-3α KO mice post-MI. Transcripts of markers of pathological hypertrophy—A, atrial natriuretic peptide (ANP); B, brain natriuretic peptide (BNP); C, Myh7 (β-myosin heavy chain)—were increased in hearts from GSK-3α KO mice as determined by quantitative PCR, 21 days post-MI. Values indicate relative expression levels in comparison with the WT sham-operated group (±SEM). All values were normalized to 18S rRNA levels. Differences between groups were determined by unpaired t test, and results are expressed as means±SEM. A probability value of ≤0.05 was considered to denote statistical significance. MI indicates myocardial infarction; WT, wild type; KO, GSK-3α(−/−); ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; PCR, polymerase chain reaction.
Figure 5
Figure 5
Deletion of GSK-3α exaggerates cardiac fibrosis, extracellular matrix remodeling, and myofibroblast activation. A, Fibrosis is increased in the KO mice post-MI. Representative images of LV remote myocardium in WT and KO stained with Masson trichrome 8 weeks post-MI. B, The percentage of LV myocardium with fibrosis was determined from 10 random images taken from each animal. C, Transcripts for collagen-1, a marker of extracellular matrix remodeling, in hearts from WT and GSK-3α KO mice were detected by quantitative PCR, 21 days post-MI. Values indicate relative expression versus WT sham-operated group (±SEM). All values were normalized to 18S rRNA levels. D, Adult fibroblasts were isolated from 3-month-old WT and GSK-3α KO mice. After 3 passages, cells were harvested and Western blotting was performed. Representative immunoblots showing increased α-SMA expression in fibroblasts from GSK-3α KO mouse hearts indicates increased myofibroblast activation in KO mouse hearts. E, Quantification of α-SMA expression in GSK-3α KO fibroblasts versus WT fibroblasts. Differences between groups were determined by unpaired t test, and results are expressed as means±SEM. A probability value of ≤0.05 was considered to denote statistical significance. MI indicates myocardial infarction; WT, wild type; KO, GSK-3α(−/−); α-SMA, a-smooth muscle actin; PCR, polymerase chain reaction.
Figure 6
Figure 6
GSK-3α deletion leads to increased post-MI apoptosis in the remote myocardium. A, Apoptosis in the remote myocardium post-MI. Two-month-old WT and GSK-3α-deficient mice were subjected to MI or sham surgery. At 3 weeks post-MI, apoptotic cells from the remote myocardium were labeled via TUNEL (red). a-SA, a myocyte-specific marker (green), was used to visualize cardiomyocytes and DAPI (blue) was used to label nuclei. Representative images of each marker are shown along with merged image. Arrows highlight a TUNEL-positive nucleus that is within the borders of a α-SA-positive cardiomyocyte. B, Quantification of rates of apoptosis is expressed as a percentage of total cells counted. Only TUNEL-positive nuclei colocalized with DAPI and within α-SA-positive cells were counted as apoptotic cardiomyocytes. At least 5000 nuclei in the remote myocardium of each animal were examined. Differences between groups were determined by unpaired t test, and results are expressed as means±SEM. A probability value of ≤0.05 was considered to denote statistical significance. TUNEL indicates terminal deoxynucleotidyl-transferase–mediated dUTP nick-end labeling; DAPI, 4,6-diamidino-2-phenylindole; α-SA, α-SArcomeric actin; MI, myocardial infarction; WT, wild type.
Figure 7
Figure 7
Deletion of GSK-3α sensitizes cardiomyocytes to ischemia and hypoxia-induced apoptosis and cellular injury. A, Apoptosis in the border zone of the infarct at 6 hours post-MI. Two-month-old WT and GSK-3α KO mice were subjected to MI. At 6 hours post-MI, heart sections were stained via TUNEL (red) and then counterstained with nuclear stain DAPI (blue). Representative images of each marker are shown along with merged image. Arrows highlight a TUNEL-positive nucleus colocalized with DAPI. B, Quantification of rates of apoptosis are expressed as a percentage of total cells. TUNEL-positive nuclei were significantly greater in GSK-3α KO mice versus WT mice. C, Cardiomyocyte apoptosis after exposure to hypoxia. Adult cardiac myocytes were isolated from 3-month-old WT and GSK-3α KO mice. Cardiomyocytes were exposed to hypoxia for 4 hours, and rates of apoptosis were quantified by TUNEL staining (red). α-SA, a myocyte-specific marker (green), was used to visualize cardiomyocytes, and DAPI (blue) was used to label nuclei. Representative images are shown along with merged image. Arrows highlight a TUNEL-positive nucleus that is within the borders of a α-SA-positive cardiomyocyte. D, Quantification of rates of apoptosis are expressed as a percentage of total cells. The number of TUNEL-positive cells was significantly greater in KO versus WT cardiomyocytes. E, As an additional measure of cell death, isolated adult mouse cardiomyocytes were exposed to hypoxia for 4 hours, and cellular toxicity was measured by the release of adenylate kinase (AK) into the cell culture medium as described in Materials and Methods. Differences between groups were determined by unpaired t test, and results are expressed as means±SEM. A probability value of ≤0.05 was considered to denote statistical significance. MI indicates myocardial infarction; WT, wild type; KO, GSK-3α(−/−); TUNEL, terminal deoxynucleotidyl-transferase–mediated dUTP nick-end labeling; DAPI, 4,6-diamidino-2-phenylindole; α-SA, α-SArcomeric actin.
Figure 8
Figure 8
Deletion of GSK-3α increased post-MI mitochondrial Bax recruitment and leakage of cytochrome C into the cytosol. A, Two-month-old WT and GSK-3α KO mice were subjected to MI or sham surgery for 3 weeks, as described in Materials and Methods. Mitochondrial and cytosolic fraction were analyzed by immunoblotting for mitochondrial Bax recruitment and cytochrome C release into the cytosol, as indicated. B through E, Bar graphs show normalized fold changes in the leakage of cytochrome C into the cytosol and Bax translocation to the mitochondria. Differences between groups were determined by unpaired t test, and results are expressed as means±SEM. A probability value of ≤0.05 was considered to denote statistical significance. MI indicates myocardial infarction; WT, wild type; KO, GSK-3α(−/−); VDAC, voltage-dependent anion channel.
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