Jody L. Martin

Small Heat Shock Proteins and Protection Against Ischemic Injury in Cardiac Myocytes

BACKGROUND Overexpression of the inducible hsp70 protects against ischemic cardiac damage. However, it is unclear whether the small heat shock proteins hsp27 and alphaB-crystallin protect against ischemic injury. METHODS AND RESULTS Our aim was to examine whether the overexpression of hsp27 and alphaB-crystallin in neonatal and adult rat cardiomyocytes would protect against ischemic injury. Recombinant adenovirus expressing hsp27 or alphaB-crystallin under the control of the cytomegalovirus promoter was used to infect cardiac myocytes at high efficiency as assessed by immunostaining. Overexpression was confirmed by Western blot analysis. Cardiomyocytes were subjected to simulated ischemic stress, and survival was estimated through assessment of lactate dehydrogenase and creatine phosphokinase release. The hsp27 overexpression decreased lactate dehydrogenase release by 45+/-7.5% in adult cardiomyocytes but had no effect in the neonatal cells. In contrast, alphaB-crystallin overexpression was associated with a decrease in cytosolic enzyme release in both adult (29+/-6.6%) and neonatal (32+/-5.4%) cardiomyocytes. Decreased endogenous hsp25 with an antisense adenovirus produced a 29+/-9.9% increase in damage with simulated ischemia. Overexpression of the inducible hsp70 in adult cardiomyocytes was associated with a 34+/-4.6% decrease in lactate dehydrogenase release and is in line with our previous results in neonatal cardiomyocytes. CONCLUSIONS The increased expression of hsp27 and alphaB-crystallin through an adenovirus vector system protects against ischemic injury in adult cardiomyocytes. Likewise, the overexpression of alphaB-crystallin protects against ischemic damage in neonatal cardiomyocytes. Decreasing the high levels of endogenous hsp25 present in neonatal cardiomyocytes renders them more susceptible to damage caused by simulated ischemia.

Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells.

Background Neural induction of human pluripotent stem cells often yields heterogeneous cell populations that can hamper quantitative and comparative analyses. There is a need for improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells (NSC), glia and neurons. One way to address this problem is to identify cell-surface signatures that enable the isolation of these cell types from heterogeneous cell populations by fluorescence activated cell sorting (FACS). Methodology/Principal Findings We performed an unbiased FACS- and image-based immunophenotyping analysis using 190 antibodies to cell surface markers on naïve human embryonic stem cells (hESC) and cell derivatives from neural differentiation cultures. From this analysis we identified prospective cell surface signatures for the isolation of NSC, glia and neurons. We isolated a population of NSC that was CD184+/CD271−/CD44−/CD24+ from neural induction cultures of hESC and human induced pluripotent stem cells (hiPSC). Sorted NSC could be propagated for many passages and could differentiate to mixed cultures of neurons and glia in vitro and in vivo. A population of neurons that was CD184−/CD44−/CD15LOW/CD24+ and a population of glia that was CD184+/CD44+ were subsequently purified from cultures of differentiating NSC. Purified neurons were viable, expressed mature and subtype-specific neuronal markers, and could fire action potentials. Purified glia were mitotic and could mature to GFAP-expressing astrocytes in vitro and in vivo. Conclusions/Significance These findings illustrate the utility of immunophenotyping screens for the identification of cell surface signatures of neural cells derived from human pluripotent stem cells. These signatures can be used for isolating highly pure populations of viable NSC, glia and neurons by FACS. The methods described here will enable downstream studies that require consistent and defined neural cell populations.

Transgene overexpression of αB crystallin confers simultaneous protection against cardiomyocyte apoptosis and necrosis during myocardial ischemia and reperfusion

We investigated whether enhanced expression of αB crystallin, a stress‐inducible molecular chaperone of the small heat shock family, can protect myocardial contractile apparatus against ischemia reperfusion (I/R) injury. Transgenic mice overexpressing αB crystallin were generated using the 0.76 kb rat αB crystallin cDNA cloned into a pCAGGS plasmid driven by a human cytomegalovirus expression system. Southern analysis confirmed transgene integration and Northern and Western blotting characterized expression (3.1‐fold and 6.9‐fold elevations in myocardial mRNA and protein levels, respectively). Extent of functional recovery overa3hreperfusion period following a 20 min ischemic period in transgenic and wild‐type mouse hearts was assessed using an ex vivo work‐performing heart preparation. The transgenic group displayed significantly higher values of DP at R45 min (29.14±1.9mmHgvs. 17.6±0.7 mm Hg), R60 min (31.56±1.7mmHgvs. 17.8±0.8 mm Hg), and R75 min (32.5±2.2 mm Hg vs. 16.9±0.9 mm Hg), and of dLVP/dt at R45 min (1740.2±111.5 mm Hg.s−1 vs. 548.7±82.2 mm Hg.s−1) and R60 min (1199.8±104.6 mm Hg.s−1 vs. 466.9±61.1 mm Hg.s−1). The transgenic group also displayed development of less oxidative stress, decreased extent of infarction, and attenuated cardiomyocyte apoptotic cell death. Transgene overexpression of αB crystallin was therefore successful in diminishing the independent contributory effects of both necrosis and apoptosis on I/R‐induced cell death.—Ray, P. S., Martin, J. L., Swanson, E. A., Otani, H., Dillmann, W. H., Das, D. K. Transgene overexpression of aB crystallin confers simultaneous protection against cardiomyocyte apoptosis and necrosis during myocardial ischemia and reperfusion. FASEB J. 15, 393‐402 (2001)

β-Catenin directly regulates Islet1 expression in cardiovascular progenitors and is required for multiple aspects of cardiogenesis

Recent studies have demonstrated that the LIM homeodomain transcription factor Islet1 (Isl1) marks pluripotent cardiovascular progenitor cells and is required for proliferation, survival, and migration of recently defined second heart field progenitors. Factors that are upstream of Isl1 in cardiovascular progenitors have not yet been defined. Here we demonstrate that β-catenin is required for Isl1 expression in cardiac progenitors, directly regulating the Isl1 promoter. Ablation of β-catenin in Isl1-expressing progenitors disrupts multiple aspects of cardiogenesis, resulting in embryonic lethality at E13. β-Catenin is also required upstream of a number of genes required for pharyngeal arch, outflow tract, and/or atrial septal morphogenesis, including Tbx2, Tbx3, Wnt11, Shh, and Pitx2. Our findings demonstrate that β-catenin signaling regulates proliferation and survival of cardiac progenitors.

The role of differential activation of p38-mitogen-activated protein kinase in preconditioned ventricular myocytes

Activation of protein kinase C (PKC) and more recently mitogen‐activated protein kinases (MAPKs) have been associated with the cardioprotective effect of ischemic preconditioning. We examined the interplay between these kinases in a characterized model of ischemic preconditioning in cultured rat neonatal ventricular cardiocytes where ectopic expression of active PKC‐δ results in protection. Two members of the MAPK family, p38 and p42/44, were activated transiently during preconditioning by brief simulated ischemia/reoxygenation. Overexpression of active PKC‐δ, rather than augmenting, completely abolished this activation. We therefore determined whether a similar process occurred during lethal prolonged simulated ischemia. In contrast to ischemia, brief, lethal‐simulated ischemia activated only p38 (2.8±0.45 vs. basal, P<0.01), which was attenuated by expression of active PKC‐δ or by preconditioning (0.48 ±0.1 vs. ischemia, P<0.01). To determine whether reduced p38 activation was the cause or an effect of protection, we used SB203580, a p38 inhibitor. SB203580 reduced ischemic injury (CK release 38.0± 3.1%, LDH release 77.3±4.0%, and MTT bioreduction 127.1 ±4.8% of control, n=20, P<0.05). To determine whether p38 activation was isoform selective, myocytes were infected with adenoviruses encoding wild‐type p38a or p38β. Transfected p38a and β show differential activation (P<0.001) during sustained simulated ischemia, with p38a remaining activated (1.48±0.36 vs. basal) but p38β deactivated (0.36± 0.1 vs. basal, P<0.01). Prior preconditioning prevented the activation of p38a (0.65±0.11 vs. ischemia, P<0.05). Moreover, cells expressing a dominant negative p38α, which prevented ischemic p38 activation, were resistant to lethal simulated ischemia (CK release 82.9±3.9% and MTT bioreduction 130.2±6.5% of control, n=8, P<0.05). Thus, inhibition of p38α activation during ischemia reduces injury and may contribute to preconditioning‐induced cardioprotection in this model.—Saurin, A. T., Martin, J. L., Heads, R. J., Foley, C., Mockridge, J. W., Wright, M. J., Wang, Y., Marber, M. S. The role of differential activation of p38‐mitogen‐activated protein kinase in preconditioned ventricular myocytes. FASEB J. 14, 2237–2246 (2000)

Overexpression of Wild-Type Heat Shock Protein 27 and a Nonphosphorylatable Heat Shock Protein 27 Mutant Protects Against Ischemia/Reperfusion Injury in a Transgenic Mouse Model

Background—The small heat shock protein 27 (hsp27) increases in expression with ischemia/reperfusion (I/R) insult in the heart. One feature of the small hsps is their ability to oligomerize and form intracellular aggregates. Oligomerization pattern is governed by the phosphorylation state of the protein that may influence their ability to protect against cellular stresses. Methods and Results—We generated transgenic (tg) mice that overexpress a wild-type human hsp27 (hsp27tg) protein or a mutant hsp27 protein (mut-hsp27tg), in which serine residues (aa15, aa78, and aa82) were replaced by alanine residues, rendering them incapable of phosphorylation. Using a Langendorff perfusion model and an intraventricular balloon, we subjected hearts to 20 minutes of ischemia followed by 1 hour of reperfusion. During reperfusion, negative and positive pressure derivatives as well as developed pressures were significantly higher in both hsp27tg and mut-hsp27tg compared with control (P<0.01) mice, with no significant difference between hsp27tg and mut-hsp27tg. Creatine kinase release during reperfusion was higher in control compared with both hsp27tg and mut-hsp27tg (P<0.05). Malondialdehyde content as well as protein oxidation products were lower in mut-hsp27tg compared with control (P<0.05). hsp27tg hearts possessed oligomers that ranged in size from small to large, whereas mut-hsp27tg hearts contained no small oligomers. Conclusions—These results indicate that in a tg mouse model, overexpression of either wild-type hsp27 or a nonphosphorylatable hsp27 mutant was equally capable of protecting the heart from I/R injury. Furthermore, the phosphorylation status of hsp27 may influence its ability to decrease oxidative stress.

Hsp27 Inhibits Bax Activation and Apoptosis via a Phosphatidylinositol 3-Kinase-dependent Mechanism

Hsp27 inhibits mitochondrial injury and apoptosis in both normal and cancer cells by an unknown mechanism. To test the hypothesis that Hsp27 decreases apoptosis by inhibiting Bax, Hsp27 expression was manipulated in renal epithelial cells before transient metabolic stress, an insult that activates Bax, induces mitochondrial injury, and causes apoptosis. Compared with control, enhanced Hsp27 expression inhibited conformational Bax activation, oligomerization, and translocation to mitochondria, reduced the leakage of both cytochrome c and apoptosis-inducing factor, and significantly improved cell survival by >50% after stress. In contrast, Hsp27 down-regulation using RNA-mediated interference promoted Bax activation, increased Bax translocation, and reduced cell survival after stress. Immunoprecipitation did not detect Hsp27-Bax interaction before, during, or after stress, suggesting that Hsp27 indirectly inhibits Bax. During stress, Hsp27 expression prevented the inactivation of Akt, a pro-survival kinase, and increased the interaction between Akt and Bax, an Akt substrate. In contrast, Hsp27 RNA-mediated interference promoted Akt inactivation during stress. Hsp27 up- or down-regulation markedly altered the activity of phosphatidylinositol 3-kinase (PI3-kinase), a major regulator of Akt. Furthermore, distinct PI3-kinase inhibitors completely abrogated the protective effect of Hsp27 expression on Akt activation, Bax inactivation, and cell survival. These data show that Hsp27 antagonizes Bax-mediated mitochondrial injury and apoptosis by promoting Akt activation via a PI3-kinase-dependent mechanism.

Ca2+/Calmodulin-Dependent Protein Kinase IIδ and Protein Kinase D Overexpression Reinforce the Histone Deacetylase 5 Redistribution in Heart Failure

Cardiac hypertrophy and heart failure (HF) are associated with reactivation of fetal cardiac genes, and class II histone deacetylases (HDACs) (eg, HDAC5) have been strongly implicated in this process. We have shown previously that inositol trisphosphate, Ca2+/calmodulin-dependent protein kinase II (CaMKII), and protein kinase (PK)D are involved in HDAC5 phosphorylation and nuclear export in normal adult ventricular myocytes and also that CaMKII&dgr; and inositol trisphosphate receptors are upregulated in HF. Here we tested whether, in our rabbit HF model, nucleocytoplasmic shuttling of HDAC5 was altered either at baseline or in response to endothelin-1, which would indicate HDAC5 phosphorylation and transcription effects. The fusion protein HDAC5–green fluorescent protein (HDAC5-GFP) was more cytosolic in HF myocytes (Fnuc/Fcyto 3.3±0.3 vs 7.2±0.4 in control), and HDAC5 was more phosphorylated. Despite this baseline cytosolic HDAC5 shift, endothelin-1 produced more rapid HDAC5-GFP nuclear export in HF versus control myocytes. We also find that PKD and CaMKII&dgr;C expression and activation state are increased in both rabbit and human HF. Inhibition of either CaMKII or PKD in HF myocytes partially restored the HDAC5-GFP Fnuc/Fcyto toward control, and simultaneous inhibition restored Fnuc/Fcyto to that in control myocytes. Moreover, adenovirus-mediated overexpression of PKD, CaMKII&dgr;B, or CaMKII&dgr;C reduced baseline HDAC5 Fnuc/Fcyto in control myocytes (3.4±0.5, 3.8±0.5, and 5.2±0.5, respectively), approaching that seen in HF. We conclude that chronic upregulation and activation of inositol trisphosphate receptors, CaMKII, and PKD in HF shifts HDAC5 out of the nucleus, derepressing transcription of hypertrophic genes. This may directly contribute to the development and/or maintenance of HF.

Diverse Mechanisms of Myocardial p38 Mitogen-Activated Protein Kinase Activation Evidence for MKK-Independent Activation by a TAB1-Associated Mechanism Contributing to Injury During Myocardial Ischemia

Abstract— The ischemic activation of p38&agr; mitogen-activated protein kinase (p38&agr;-MAPK) is thought to contribute to myocardial injury. Under other circumstances, activation is through dual phosphorylation by MAPK kinase 3 (MKK3). Therefore, the mkk3−/− murine heart should be protected during ischemia. In retrogradely perfused mkk3−/− and mkk3+/+ mouse hearts subjected to 30 minutes of global ischemia and 120 minutes of reperfusion, infarction/risk volume was similar (50±5 versus 51±4, P =0.93, respectively), as was intraischemic p38-MAPK phosphorylation (10 minutes ischemia as percent basal, 608±224 versus 384±104, P =0.43, respectively). This occurred despite undetectable activation of MKK3/6 in mkk3−/− hearts. However, tumor necrosis factor (TNF)-induced p38-MAPK phosphorylation was markedly diminished in mkk3−/− vs mkk3+/+ hearts (percent basal, 127±23 versus 540±267, respectively, P =0.04), suggesting an MKK-independent activation mechanism by ischemia. Hence, we examined p38-MAPK activation by TAB1-associated autophosphorylation. In wild-type mice and mkk3−/− mice, the p38-MAPK catalytic site inhibitor SB203580 (1 &mgr;mol/L) diminished phosphorylation during ischemia versus control (10 minutes ischemia as percent basal, 143±2 versus 436±96, P =0.003, and 122±25 versus 623±176, P =0.05, respectively) and reduced infarction volume (infarction/risk volume, 57±5 versus 36±3, P <0.001, and 50±5 versus 29±3, P =0.003, respectively) but did not alter TNF-induced activation, although in homogenates of ischemic hearts but not TNF-exposed hearts, p38-MAPK was associated with TAB1. Furthermore, adenovirally expressed wild-type and drug-resistant p38&agr;-MAPK, lacking the SB203580 binding site, was phosphorylated when H9c2 myoblasts were subjected to simulated ischemia. However, SB203580 (1 &mgr;mol/L) did not prevent the phosphorylation of resistant p38&agr;-MAPK. These findings suggest the ischemic activation of p38-MAPK contributing to myocardial injury is by TAB1-associated autophosphorylation.

Inhibition of Phosphatidylinositol 3-Kinase Activity by Adenovirus-mediated Gene Transfer and Its Effect on Insulin Action

Phosphatidylinositol 3-kinase (PI 3-K) is implicated in cellular events including glucose transport, glycogen synthesis, and protein synthesis. It is activated in insulin-stimulated cells by binding of the Src homology 2 (SH2) domains in its 85-kDa regulatory subunit to insulin receptor substrate-1 (IRS-1), and, others. We have previously shown that IRS-1-associated PI 3-kinase activity is not essential for insulin-stimulated glucose transport in 3T3-L1 adipocytes, and that alternate pathways exist in these cells. We now show that adenovirus-mediated overexpression of the p85N-SH2 domain in these cells behaves in a dominant-negative manner, interfering with complex formation between endogenous PI 3-K and its SH2 binding targets. This not only inhibited insulin-stimulated IRS-1-associated PI 3-kinase activity, but also completely blocked anti-phosphotyrosine-associated PI 3-kinase activity, which would include the non-IRS-1-associated activity. This resulted in inhibition of insulin-stimulated glucose transport, glycogen synthase activity and DNA synthesis. Further, Ser/Thr phosphorylation of downstream molecules Akt and p70 S6 kinase was inhibited. However, co-expression of a membrane-targeted p110C AAX with the p85N-SH2 protein rescued glucose transport, supporting our argument that the p85N-SH2 protein specifically blocks insulin-mediated PI 3-kinase activity, and, that the signaling pathways downstream of PI 3-kinase are intact. Unexpectedly, GTP-bound Ras was elevated in the basal state. Since p85 is known to interact with GTPase-activating protein in 3T3-L1 adipocytes, the overexpressed p85N-SH2 peptide could titrate out cellular GTPase-activating protein by direct association, such that it is unavailable to hydrolyze GTP-bound Ras. However, insulin-induced mitogen-activated protein kinase phosphorylation was inhibited. Thus, PI 3-kinase may be required for this action at a step independent of and downstream of Ras. We conclude that, in 3T3-L1 adipocytes, non-IRS-1-associated PI 3-kinase activity is crucial for insulin’s metabolic signaling, and that overexpressed p85N-SH2 protein inhibits a variety of insulin’s ultimate biological effects.