We examined whether cellular antioxidant-defence enhancement preserves diastolic dysfunction via regulation of both diastolic intracellular free Zn2+ and Ca2+ levels ([Zn2+]i and [Ca2+]i) levels N-acetyl cysteine (NAC) treatment (4 weeks) of diabetic rats preserved altered cellular redox state and also prevented diabetes-induced tissue damage and diastolic dysfunction with marked normalizations in the resting [Zn2+]i and [Ca2+]i. The kinetic parameters of transient changes in Zn2+ and Ca2+ under electrical stimulation and the spatiotemporal properties of Zn2+ and Ca2+ sparks in resting cells are found to be normal in the treated diabetic group. Biochemical analysis demonstrated that the NAC treatment also antagonized hyperphosphorylation of cardiac ryanodine receptors (RyR2) and significantly restored depleted protein levels of both RyR2 and calstabin2. Incubation of cardiomyocytes with 10 µM ZnCl2 exerted hyperphosphorylation in RyR2 as well as higher phosphorphorylations in both PKA and CaMKII in a concentration-dependent manner, similar to hyperglycemia. Our present data also showed that a subcellular oxidative stress marker, NF-κB, can be activated if the cells are exposed directly to Zn2+. We thus for the first time report that an enhancement of antioxidant defence in diabetics via directly targeting heart seems to prevent diastolic dysfunction due to modulation of RyR2 macromolecular-complex thereby leading to normalized [Ca2+]i and [Zn2+]i in cardiomyocytes.

Current data support that pharmacological modulators of endoplasmic reticulum stress (ERS) have therapeutic potential for diabetic individuals. Therefore, we aimed to examine whether timolol, having free radical-scavenger action, besides being a β-blocker, exerts a cardioprotective effect via inhibition of ERS response in diabetic rats in a comparison with an antioxidant N-acetylcysteine (NAC). Histopathological data showed that either timolol- or NAC-treatment of diabetic rats prevented the changes in mitochondria and nucleus of the cardiac tissue while they enhanced the cellular redox-state in heart as well. The levels of ER-targeted cytoprotective chaperones GRP78 and calnexin, unfolded protein response signaling protein CHO/Gadd153 besides the levels of calpain, BCL-2, phospho-Akt, PUMA, and PML in the hearts from diabetic rats, treated with either timolol or NAC, are found to be similar among these groups, although all these parameters were markedly preserved in the untreated diabetics compared to those of the controls. Taken into consideration how important a balanced-ratio between anti-apoptotic and pro-apoptotic proteins for the maintenance mitochondria/ER function, our results suggest that ERS in diabetic rat heart is mediated by increased oxidative damage, which in turn triggers cardiac dysfunction. Moreover, we also demonstrated that timolol treatment of diabetic rats, similar to NAC treatment, induced a well-controlled redox-state and apoptosis in cardiac myocardium. We, thus for the first time, report that cardioprotective effect of timolol seems to be associated with normalization of ER function due to its antioxidant action in cardiomyocytes even under hyperglycemia.

Trace elements can participate in the catalysis of group-transfer reactions and can serve as their structural components. However, most of them including zinc and selenium have multifunctional roles in biological environments such as antioxidant and/or pro-oxidant effects, as concentration-dependent manner. Although it has been demonstrated the antioxidant actions of either selenium or zinc compounds, there are several documents pointing out their pro-oxidant/oxidant roles in biological systems. Here we have used ELISA-based immuno-spin trapping, a method for detection of free radical formation, to detect whether or not a zinc compound, Zn3(PO4)2, or a selenium compound, Na2SeO3, has antioxidant and/or pro-oxidant effect on 5,5-Dimethyl-1-Pyrroline-N-Oxide (DMPO)-DNA nitrone adducts induced with Cu(II)-H2O2-oxidizing system in in vitro preparations. Second, we examined whether this technique is capable to demonstrate the different DMPO-protein nitrone adduct productions in isolated protein crude of hearts from normal rats (CON) or rats with metabolic syndrome (MetS). Our data demonstrated that either Zn(2+) (100 µM) or SeO3(-2) (50 nM) has very strong antioxidant action against 200 µM H2O2-induced DMPO-DNA nitrone adduct production, whereas their higher concentrations have apparent pro-oxidant actions. We also used verification by Western blotting analysis whether immuno-spin trapping can be used to assess H2O2-induced DMPO-protein nitrone adducts in heart protein crudes. Our Western blot data further confirmed the ELISA-data from proteins and demonstrated how Zn(2+) or SeO3(-2) are dual-functioning ions such as antioxidant at lower concentrations while pro-oxidant at higher concentrations. Particularly, our present data with SeO3(-2) in DMPO-protein nitrone adducts, being in line with our previous observation on its dual-actions in ischemia/reperfusion-induced damaged heart, have shown that this ion has higher pro-oxidant actions over 50 nM in MetS-group compared to that of CON group.

Intracellular free Zn 2þ concentration, (Zn 2þ) i is less than one nanomolar in resting cardiomyocytes. Very little is known about mechanisms controlling (Zn 2þ) i distribution and variations during cardiac function although in vitro oxidant exposures to cardiomyocytes caused 30-fold increase in (Zn 2þ) i but only 2-fold in (Ca 2þ) i while these were 2-fold and 1.2-fold under hyperglycemia, respectively. Since it has been mentioned that Zn 2þ homeostasis is involved in unfolded protein response (endoplasmic reticulum stress, ERS) under salt stress, we aimed to investigate whether a hyperglycemia-induced increased level of basal (Zn 2þ) i can trigger an ERS, which in turn induces marked cardiomyocyte dysfunction. When we enforced Zn 2þ influx into cytosol by using zinc ionophore pyrithione (ZnPT), mechanical and electrical activities of cardiomyocytes impaired in a concentration-dependent manner, similar to hyperglycemia. Incubation of cardiomyocytes with ZnPT exerted also hyperphosphorylation in RyR2, PKA, and CaMKII in a concentration-dependent manner, again similar to hyperglycemia. The levels of some ERS chaperon proteins and ERSmediated apoptosis marker proteins (Calregulin, GRP 78, Bcl-2 and PUMA) were also examined and compared in between ZnPT-incubated cardiomyocytes and STZ-diabetic rat cardiomyocytes. The chaperon protein levels were found to be increased in diabetics and also in ZnPT-incubated ones in a concentration dependent-manner. We also observed a similar parallel correlation between these two-group experiments for anti-apoptotic protein Bcl-2 and apoptotic one PUMA. Taken into consideration diabetes-induced cardiac dysfunction due to, in part, increased basal (Zn 2þ) i and in vitro increased (Zn 2þ) i-induced cardiomyocyte dysfunction present a possible triggering role of increased (Zn 2þ) i in hyperglycemia-induced cardiomyocyte dysfunction through ERS.

Mitochondrial Ca 2þ regulates a wide variety of physiological processes, including ATP production and cell death. Rapid Ca 2þ uptake into mitochondria is mediated by the mitochondrial calcium uniporter (MCU) complex, which is composed of the pore forming MCU protein, and the regulatory proteins EMRE, and MICU1/MICU2. Currently, the submitochondrial localization and transmembrane orientation of these proteins are either unknown or under debate. We attack these issues using a classical thiol-modification approach. We removed native MCU complex proteins in HEK293 cells using CRISPR/Cas9, and reintroduced mutants containing a single cysteine at defined positions. Treatments of the mitoplast with a bulky, thiol-reactive compound polyethylene glycol (PEG) maleimide would alter the molecular weight of the protein if the engineered Cys is exposed to the intermembrane space. Using this strategy along with other biochemical methods, we demonstrated that (1) MCU adopts an orientation with the signature DIME motif facing intermembrane space (IMS), ( 2) the conserved C-terminal polyaspartic tail of EMRE is in the IMS, (3) both MICU1 and MICU2 are associated with the outer leaflet of the inner membrane, and (4) although MICU1 is a peripheral membrane protein, MICU2 is an integral component of the inner membrane. Then, applying domain interaction analysis and mutagenesis screening, we identified molecular contacts that govern the Ca 2þ transport behavior of the MCU complex. In particular, we demonstrate that EMRE interacts with MCU through the transmembrane helices to activate the Ca 2þ pore, while using the polyaspartic tail to recruit MICU1/2 to gate the pore. This dual functionality of EMRE ensures that all functional MCU complexes respond appropriately to Ca 2þ stimuli from the cytosol, safeguarding against dangerous Ca 2þ leakage, which could diminish mitochondrial energy output and potentially trigger apoptotic cell death.