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Overexpression of the stress protein Grp94 reduces cardiomyocyte necrosis due to calcium overload and simulated ischemia¹

MAURIZIO VITADELLO², DANIELE PENZO^*,2, VALERIA PETRONILLI, GENNY MICHIELI^*, SELENA GOMIRATO^*, ROBERTA MENABO‘, FABIO DI LISA and LUISA GORZA^*,3

CNR Institute of Neuroscience, and Departments of
^* Biomedical Sciences and
Biochemistry, University of Padova, Padova, Italy

³Correspondence: Department of Biomedical Sciences, via G. Colombo 3, 35121, Padova, Italy. E-mail: lgorza{at}bio.unipd.it

SPECIFIC AIMS

Stress proteins have been demonstrated to be involved in myocardial protection against ischemic injury; however, the precise mechanism(s) whereby their protective role is exerted remains to be elucidated. To investigate whether and by which mechanism the sarcoplasmic reticulum stress protein Grp94 is involved in myocyte protection against calcium overload and simulated ischemia, we performed cytoprotection studies using either stably transfected myogenic cell lines or transiently transduced neonatal rat cardiomyocytes that overexpressed Grp94.

PRINCIPAL FINDINGS

1. Grp94 overexpression in stably transfected myogenic cell lines
Western blot analysis using anti-Grp94 monoclonal antibodies showed a three- to eightfold increase in Grp94 total amount in four different murine skeletal C2C12 cell clones and in one rat embryonic cardiac H9c2 clone, stably transfected with grp94 cDNA, compared with control clones. Parallel analyses of Grp78 and calreticulin expression did not reveal significant changes in the amount of these proteins in most Grp94-overexpressing clones, and no expression of inducible Hsp70 was observed in any control or overexpressing clone.

2. Extent of cell necrosis after calcium overload of Grp94-overexpressing C2C12 clones
After 45 min exposure to 1, 2.5, or 10 µmol/L of the calcium ionophore A23187, Grp94-overexpressing C2C12 cells released significantly less LDH than cells from the control clones (P<0.001). Time course analysis showed that LDH released from Grp94-overexpressing cells corresponded to 63%, 70%, and 80% of LDH released by cells of the control clone 45, 60, and 90 min after exposure to 2.5 µmol/L A23187, respectively (P<0.001). Increased resistance to necrosis (also revealed by reduced propidium iodide uptake) was observed for all the Grp94-overexpressing clones.

3. Grp94 overexpression and calcium homeostasis
Free intracellular calcium concentration [Ca²⁺]_i was monitored during exposure to the ionophore of control and Grp94-overexpressing C2C12 clones (Fig. 1 A). In control cells, addition of A23187 induced a prompt rise in [Ca²⁺]_i, which saturated fluo3 fluorescence within 1 min. At variance, Grp94-overexpressing cells showed a more gradual increase in [Ca²⁺]_i, which remained significantly lower than in control clones during the first 10 min after A23187 addition. Evaluated in the same experiments after 20 min of ionophore exposure, propidium iodide uptake occurred in a significantly larger number of control cells than Grp94-overexpressing cells (Fig. 1B ). Exposure for 45 min to 2 µmol/L thapsigargin and 2.5 µmol/L ionophore A23187 did not affect resistance of Grp94-overexpressing cells to necrotic death: similar to what occurs in the presence of the ionophore alone, they displayed a significantly reduced release of LDH activity compared with control clone cells (mean and SD: 7.89±0.91% and 23.31±1.02%, respectively, n=10, P<0.0001).

View larger version (17K): [in this window] [in a new window]   Figure 1. Kinetic of [Ca 2+]i in control and Grp94-overexpressing clones after A23187addition. A) Fluo-3 fluorescence ratio of control clone S1H2 and Grp94-overexpressing clone 84E5 after exposure to A23187. Values represent mean ± SD from 3 different experiments. Asterisks indicate significant difference between values of control and overexpressing clones at the same time point. B) Percentage of propidium iodide-positive cells observed in control clone S1H2 (striped bar) and Grp94-overexpressing clone 84E5 (black bar) after 20 min of exposure to A23187 and [Ca 2+]ikinetic analysis. Values represent mean ± SE from 3 independent experiments; n = total number of cells counted.

4. Extent of ionophore-induced necrosis in Grp94-overexpressing cardiac cells
Additional experiments were performed using stably transfected cells obtained from the H9c2 cardiac muscle cell line and transiently transfected rat neonatal cardiomyocytes. Grp94-overexpressing H9c2 cells exposed to 2.5 µM A23187 for 65 min showed a signifi-cant reduction in released LDH activity compared with the control clone (P<0.001). Primary cultures of newborn rat ventricular myocardium were induced to overexpress Grp94 by transient transfection. Transduced myocytes were identified with immunofluorescence for the concomitant expression of GFP, and necrotic death was visualized by the presence of red nuclear fluorescence due to propidium iodide uptake. Upon treatment with 5 µM A23187 for 30 min at 37°C, cardiomy-ocytes overexpressing Grp94 showed a reduced number of nuclei labeled with propidium iodide than myocytes transfected with control construct only (14.0% and 6.6%, respectively).

5. Extent of necrosis after simulated ischemia of Grp94-overexpressing cardiac cells
Simulated ischemia was performed exposing cells to ischemic buffer (118 mmol/L NaCl, 24 mmol/L NaHCO₃, 1 mmol/L NaH₂PO₄H₂O, 1.2 mmol/L MgCl₂, 2.5 mmol/L CaCl₂2H₂O, 0.5 mmol/L NaEDTA2H₂O, 20 mmol/L sodium lactate, and 16 mmol/L KCl, pH 6.2) in an ischemia chamber and maintained at 37°C in 100% N₂. After 16 h, Grp94-overexpressing H9c2 clone displayed a significantly lower percentage of necrotic cells (evaluated using propidium iodide uptake) than the control clone (Fig. 2 A). A comparable result was observed when transiently transfected primary cardiomyocytes were exposed to 4 h of simulated ischemia: only 8.5% of Grp94-overexpressing/GFP-positive cardiomyocytes were necrotic/propidium iodide positive vs. 17.7% of control/GFP-positive cardiomyocytes (Fig. 2B ).

View larger version (18K): [in this window] [in a new window]   Figure 2. Effect of simulated ischemia on Grp94-overexpressing H9c2 cells and neonatal ventricular cardiomyocytes. A) Percentage of propidium iodide-positive cells observed in control clone SH-2B1 (striped bar) and Grp94-overexpressing clone 8H-5G8 (black bar) after 16 h normoxia in growth medium, normoxia in the presence of ischemic buffer (IB) and anoxia in the presence of IB (simulated ischemia). Values represent mean ± SE of 3 or 4 independent experiments; n, total number of cells evaluated. Asterisk indicates the presence of significant difference between the percentage of necrotic cells of Grp94-overexpressing and control clones after exposure to simulated ischemia (P<0.01). B) Percentage of GFP- and propidium iodide-positive myocytes observed after exposure to 4 h of simulated ischemia of primary cultures of neonatal ventricular cardiomyocytes, transfected with pT or pT94 constructs. n indicates the total number of GFP-positive ventricular myocytes observed in several slides from 2 independent experiments. Note the lower percentage of necrotic cardiomyocytes in the presence of Grp94 overexpression.

CONCLUSIONS AND SIGNIFICANCE

Several studies reported a significant increase in Grp94 levels after exposure of different cell types to mild ischemia and to other conditions known to trigger delayed cytoprotection; other elegant studies demonstrated that reduced levels of Grp94 or lack of stress-induced increase of Grp94 were accompanied by reduced cell viability. To our knowledge, no investigation so far has addressed the effects of Grp94 overexpression and its mechanistic involvement in cardiomyocyte resistance against calcium overload or ischemic death.

We previously showed that in atrial myocytes of goats affected by chronic atrial fibrillation, a pathology characterized by early signs of calcium overload, the increase in Grp94 expression occurs concomitant with a significant reduction in membrane-bound calcium deposits, which we interpreted it as a potentially protective response. Here we demonstrate that increased levels of Grp94 reduced necrotic death of rat neonatal cardiomyocytes exposed to calcium overload. Parallel investigations in myogenic clones with stable Grp94 overexpression allowed us not only to exclude the notion that significant changes in expression of Grp78 or calreticulin may contribute to enhanced resistance to cell necrosis, but also to unravel the contribution of Grp94 as a putative calcium binding protein. The reduced rate of [Ca²⁺]_i increase of Grp94-overexpressing cells exposed to the ionophore supports the ability of this protein to act (although to a limited extent) as an efficacious Ca²⁺ buffer. Such a delay in [Ca²⁺]_i increase is responsible for the reduced extent of necrosis revealed by propidium iodide uptake, which precedes the difference in LDH release observed between control and Grp94-overexpressing clones. The mechanism through which Grp94 exerts calcium buffering remains elusive. Experiments performed in the presence of ionophore and the ER calcium pump inhibitor thapsigargin show that cytoprotection is still detectable, suggesting that Grp94 maintains cell viability retaining calcium in the ER. However, this does not rule out the possibility that some Grp94 molecules displaying transmembrane localization can participate to calcium homeostasis via a cytoplasmic domain.

More relevant to pathophysiology and the maintenance of calcium homeostasis in the living cells, where changes in [Ca²⁺]_i rarely transcend the micromolar range, are results obtained in the presence of simulated ischemia, where Grp94 overexpression increases resistance against necrosis of cardiac H9c2 cells and primary cardiomyocytes. Although the experimental approach used (i.e., exposure to anoxia in the presence of ischemic buffer) hampered [Ca ²⁺]_i monitoring, several mechanisms interact to deregulate [Ca ²⁺]_i in this condition. The participation of Grp94 to calcium homeostasis has been overlooked so far due to the prevailing attention to other ER calcium binding proteins. Our evidence for a role in the maintenance of calcium homeostasis for a constitutive yet inducible protein like Grp94 might be relevant in understanding self-defense mechanisms operating in injured cardiomyocytes, as well as for developing novel therapeutic strategies to increase their resistance against ischemia.

View larger version (14K): [in this window] [in a new window]   Figure 3. Schematic diagram illustrating Grp94 involvement in calcium homeostasis. Increased Grp94 levels delay increase in [Ca 2+]i ; in turn, ER stresses comprising those characterized by [Ca 2+]i increase up-regulate Grp94 expression.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0644fje; to cite this article, use FASEB J. (March 28, 2003) 10.1096/fj.02-0644fje

² Both authors contributed equally to this work.

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