Loss of Adult Cardiac Myocyte GSK-3 Leads to Mitotic Catastrophe Resulting in Fatal Dilated Cardiomyopathy

Abstract

Rationale: Cardiac myocyte-specific deletion of either glycogen synthase kinase (GSK)-3α and GSK-3β leads to cardiac protection after myocardial infarction, suggesting that deletion of both isoforms may provide synergistic protection. This is an important consideration because of the fact that all GSK-3-targeted drugs, including the drugs already in clinical trial target both isoforms of GSK-3, and none are isoform specific.

Objective: To identify the consequences of combined deletion of cardiac myocyte GSK-3α and GSK-3β in heart function.

Methods and results: We generated tamoxifen-inducible cardiac myocyte-specific mice lacking both GSK-3 isoforms (double knockout). We unexpectedly found that cardiac myocyte GSK-3 is essential for cardiac homeostasis and overall survival. Serial echocardiographic analysis reveals that within 2 weeks of tamoxifen treatment, double-knockout hearts leads to excessive dilatative remodeling and ventricular dysfunction. Further experimentation with isolated adult cardiac myocytes and fibroblasts from double-knockout implicated cardiac myocytes intrinsic factors responsible for observed phenotype. Mechanistically, loss of GSK-3 in adult cardiac myocytes resulted in induction of mitotic catastrophe, a previously unreported event in cardiac myocytes. Double-knockout cardiac myocytes showed cell cycle progression resulting in increased DNA content and multinucleation. However, increased cell cycle activity was rivaled by marked activation of DNA damage, cell cycle checkpoint activation, and mitotic catastrophe-induced apoptotic cell death. Importantly, mitotic catastrophe was also confirmed in isolated adult cardiac myocytes.

Conclusions: Together, our findings suggest that cardiac myocyte GSK-3 is required to maintain normal cardiac homeostasis, and its loss is incompatible with life because of cell cycle dysregulation that ultimately results in a severe fatal dilated cardiomyopathy.

Keywords: GSK-3; cell cycle; dilated cardiomyopathy; heart failure; mitotic catastrophe.

Figure 1. Cardiac myocyte-specific deletion of GSK3 leads to severe cardiac dysfunction and death

A, Diagrammatic representation of the timeline for tamoxifen (tam)-induced Cre-recombinase mediated deletion of GSK3. B, Representative Western blot showing tamoxifen-induced deletion of GSK-3α/β (n=4). C, Kaplan-Meier survival curves for DKO mice versus controls indicate a significant reduction in lifespan within 40 days of tam-treatment. D–G, WT and DKO mice underwent baseline transthoracic echocardiographic examination and subjected to tamoxifen protocol. Mice were then followed with serial echocardiography at the time points shown. D, Left ventricular internal dimension at end-diastole (LVID;d). E, LVID at end-systole (LVID;s). F, left ventricular ejection fraction (LVEF). G, LV fractional shortening (LVFS). * P<0.05; ***P<0.005. Tam, tamoxifen.

Figure 2. Cardiac myocyte-specific deletion of GSK3 leads to dilated cardiomyopathy, cardiac myocyte enlargement, accelerated fibrosis and heart failure

A, Gross morphology of hearts from DKO versus control demonstrates multi-chamber enlargement. Representative images displaying morphological changes in a temporal manner. B, Cardiac myocyte cross-sectional area was significantly increased in the DKO hearts at d21 of tamoxifen timeline (n=4). C, Morphometric analysis of cardiac hypertrophy in d25 animals using heart weight/tibia length (HW/TL) ratio indicates significant increases in HW/TL ratio in DKO mice (n=17) compared to controls (n=21). D, Representative Trichrome stained heart sections E, quantification of fibrosis demonstrating increased fibrosis in DKO hearts starting from day 21 at tamoxifen timeline. F, Representative H&E stained lung sections demonstrate thickening of alveolar interstitium (arrow). ***P<0.005.

Figure 3. DKO mice demonstrate ultra-structural defects and enlarged cardiac myocyte nuclei

A, Representative H&E stained heart sections from DKO versus control mice on d35 of the tam-timeline showing enlarged nuclei in multiple chambers (Arrows indicate enlarged cardiac myocyte nuclei, B.C indicates blood clot). B, Representative transmission electron micrographs of d24 mouse hearts on tam-timeline demonstrate widened sarcomere z-line (arrow) and disrupted mitochondrial morphology (solid triangle). C&D, Representative transmission electron micrographs of d24 mouse hearts on tam-timeline demonstrate enlarged nuclei (Nu) with nuclear aggregates (NO), abnormal sarcomeres (mf), and nuclear membrane invaginations.

Figure 4. DKO leads to activation of cell cycle and apoptosis in cardiac myocytes but does not in fibroblasts

A, At 2 wk of tamoxifen timeline, cardiac myocytes and fibroblasts were isolated from DKO and littermate controls hearts and lysates were analyzed by western blotting. B, Quantification of α-smooth muscle actin (α-SMA) shows unaltered myofibroblast activation. C–E, Western blot analysis reveals activation of cell cycle pathways and apoptosis specifically in cardiac myocytes only. **P<0.005.

Figure 5. GSK3 deletion induces cell cycle re-entry with polyploidization and multinucleation

A, Representative H&E images of DKO left ventricle from d35 mice on the tam-timeline reveal the presence of multi-lobulated (box) and multinucleated cardiac myocytes (arrow). B, Quantitation of BrdU+/ α-actinin+ cardiac myocytes from d24 mice on the tam-timeline reveals statistically significant increases in DNA synthesis in DKO (n=3) cardiac myocytes compared to control (n=3). C, Flow cytometry analysis of PCM-1+ nuclei from isolated adult d21 cardiac myocytes on the tam-timeline. Results indicate a statistically significant increase in nuclei with >4N in the DKO (n=3). D, Representative immunofluorescence images of adult d21 cardiac myocytes mice on the tam-timeline stained with α-actinin and DAPI revealed cardiac myocytes with up to 4 nuclei in karyokinesis in the DKO compared to control. E, Quantitation of number of DAPI+ nuclei in isolated adult cardiac myocytes from d24 mice on the tam-timeline reveals a statistically significant reduction in bi-nucleated cardiac myocytes and increase in cardiac myocytes with greater than 4 nuclei in DKO (n= 493 cells from 5 mice) versus controls (n = 386 cells from 3 mice).

Figure 6. DKO cardiac myocytes show mitotic entry and DNA damage

A, Representative Western blot analyses of various markers of the cell cycle. Results indicate increased protein expression of CDK1, Cyclin B1, and CDC25C pSer-216 in DKO hearts compared to controls. B, Graph showing folds changes in CDK1, Cyclin B1 and CDC25C (pSer-216). C, Representative Western blots of DNA damage and cell cycle checkpoint markers γ-H2A.X, CHK2 (pThr68), p21 and p27. D, Graph showing fold changes in γ-H2A.X and -CHK2 (pThr68), p21 and p27 in the DKO hearts compared to controls. E, Representative immunofluorescence staining for γ-H2A.X in isolated adult cardiac myocytes from d25 mice on the tam-timeline shows predominant detection of γ-H2A.X+ nuclei in DKO cardiac myocytes (n=3). F, Representative immunofluorescence staining for pH3-Ser10 in cardiac sections from d21 mice on the tam-timeline shows predominant detection in α-actinin/ pH3-Ser10 dual positive nuclei in DKO cardiac myocytes. G, Quantitation of pH3-Ser10 (red) in α-actinin (green)/DAPI+ (blue) nuclei shows statistically significant increase in DKO cardiac myocytes (n=3029 nuclei from 3 mice) compared to controls (n=3680 nuclei from 3 mice). *P<0.05; ***P<0.005.

Figure 7. Mitotic entry of DKO cardiac myocytes leads to death by mitotic catastrophy

A, Quantitative TUNEL analysis on cardiac sections from d35 mice on the tam-timeline show a significant increase in TUNEL-positive cardiac myocytes in the DKO (n= 6513 cells from 6 mice) compared to controls (n= 5211 cells from 5 mice). B, Representative Western blots of apoptotic markers indicate a steady increase in p53 and an decrease in BCL-2/BAX ratio after tam-administration in DKO hearts compared to controls (n=4). C, Graphical representation of the ratio of Bcl-2/Bax in DKO hearts to control hearts during the Tam-timeline. Results indicate a significant reduction in the anti-apoptotic Bcl-2 to pro-apoptotic Bax protein ratio (n=4). *P<0.05; ***P<0.005. D, Representative immunofluorescence staining for pH3-Ser10/TUNEL dual positive nuclei from cardiac sections at d21 mice on the tam-timeline shows predominant detection of dual positive nuclei in DKO hearts. E, Quantitation of the distribution of pH3+/TUNEL+/DAPI+ nuclei in the DKO versus control heart sections. Results indicate a significant proportion of triple+ nuclei in the DKO versus controls. P value <.05, Chi-Square test.

Figure 8. Schematic representation of the effects of GSK3 deletion on cardiac myocyte cell cycle

During normal cardiac development cardiac myocytes retain the ability to proliferate. These cells are mono-nucleated during early development to birth but progress to a predominantly post-mitotic, bi-nucleated state by maturity. Upon deletion of GSK3, adult cardiac myocytes re-enter cell cycle. Cell cycle re-entry is opposed by cell cycle checkpoint activation and DNA damage. Although it is clear that DKO cardiac myocytes are able to bypass critical checkpoints to complete karyokinesis, these cells have impaired mitotic capacity and do not progress to cytokinesis. Instead, abnormal mitosis within these cells induces mitotic catastrophe resulting in a loss of functional cardiac myocytes. A possible alternative hypothesis showing loss of cardiac myocyte GSK-3 may lead to apoptotic cell death without cell cycle reentry has been shown by dotted line. Mice develop dilated cardiomyopathy as a result of cardiac myocyte loss and ultimately succumb to death.