The signalling pathway of CaMKII-mediated apoptosis and necrosis in the ischemia/reperfusion injury

Abstract

Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) plays an important role mediating apoptosis/necrosis during ischemia-reperfusion (IR). We explored the mechanisms of this deleterious effect. Langendorff perfused rat and transgenic mice hearts with CaMKII inhibition targeted to sarcoplasmic reticulum (SR-AIP) were subjected to global IR. The onset of reperfusion increased the phosphorylation of Thr(17) site of phospholamban, without changes in total protein, consistent with an increase in CaMKII activity. Instead, there was a proportional decrease in the phosphorylation of Ser2815 site of ryanodine receptors (RyR2) and the amount of RyR2 at the onset of reperfusion, i.e. the ratio Ser2815/RyR2 did not change. Inhibition of the reverse Na(+)/Ca(2+)exchanger (NCX) mode (KBR7943) diminished phospholamban phosphorylation, reduced apoptosis/necrosis and enhanced mechanical recovery. CaMKII-inhibition (KN-93), significantly decreased phospholamban phosphorylation, infarct area, lactate dehydrogenase release (LDH) (necrosis), TUNEL positive nuclei, caspase-3 activity, Bax/Bcl-2 ratio and Ca(2+)-induced mitochondrial swelling (apoptosis), and increased contractile recovery when compared with non-treated IR hearts or IR hearts pretreated with the inactive analog, KN-92. Blocking SR Ca(2+) loading and release (thapsigargin/dantrolene), mitochondrial Ca(2+) uniporter (ruthenium red/RU360), or mitochondrial permeability transition pore (cyclosporine A), significantly decreased infarct size, LDH release and apoptosis. SR-AIP hearts failed to show an increase in the phosphorylation of Thr(17) of phospholamban at the onset of reflow and exhibited a significant decrease in infarct size, apoptosis and necrosis respect to controls. The results reveal an apoptotic-necrotic pathway mediated by CaMKII-dependent phosphorylations at the SR, which involves the reverse NCX mode and the mitochondria as trigger and end effectors, respectively, of the cascade.

Figure 1. CaMKII inhibition increased contractile recovery and diminished necrosis and apoptosis in IR

A. Left panel: Recovery of left ventricular developed pressure (LVDP) in hearts submitted to the IR (45/120min) protocol, in the presence and absence of CaMKII-inhibition (2.5μM KN-93 administrated 10min before ischemia and during the first 10min of reperfusion. n=4 hearts in each group. Right panel: KN significantly reduced infarct size and LDH release in hearts submitted to IR, indicating the prevention of IR-induced necrosis, n= 5–10 experiments. B. 2.5μM KN significantly reduced TUNEL positive cells (brown nuclei with arrow) after IR compared to hearts without KN-93. Bar represents 40μm. The bar graph on the right panel shows pooled data from these experiments, n=3–7. C. CaMKII-inhibition reduced Bax/Bcl-2 ratio and caspase-3 activity supporting the participation of CaMKII in the apoptotic pathway; n=4–5 experiments. *P<0.05 vs. IR in the absence of KN. D. Total PLN and phosphorylation of Thr¹⁷ site at 1–3 min of reperfusion; n= 5–9 experiments. *P<0.05 vs. preischemia (preisch).

Figure 2

A. CaMKII is activated by NCX-induced Ca²⁺ influx. KBR (5μM), but not nifedipine (0.4μM) prevented the CaMKII-dependent phosphorylation of Thr¹⁷ of PLN in hearts submitted to IR (left panel). NCX inhibition also significantly improved LVDP recovery (right panel). B. Inhibition of reverse NCX mode protected hearts submitted to IR from cell death. KBR-treated hearts also showed diminished infarct size and LDH release (left panel) and a decrease in the number of apoptotic cells (right panel). n=4–5 experiments * P<0.05 vs. IR.

Figure 3. The SR participates in myocyte death induced by IR

A. Inhibition of SERCA2a with thapsigargin (Thaps) and RyR2 with dantrolene (Dant) diminished the infarct size. Above: typical TTC staining for each situation; below: average of 4–5 experiments. B. Thaps and Dant significantly diminished apoptosis. Representative microphotographies of TUNEL stained sections. Bar represents 40μm. Below to the right: average values of 4–5 experiments. * P<0.05 vs. IR.

Figure 4. The SR-CaMKII-dependent phosphorylations participate in IR injury

(A) Increase in phosphorylation of Thr¹⁷ site of PLN at the onset of reperfusion (3min) in WT mice. This increase was not apparent in SR-AIP mice with CaMKII inhibition targeted to the SR. SR-AIP showed a significant decrease in infarct size (B), LDH release (C), TUNEL-positive cells (D), and Bax/Bcl-2 ratio (E). Bar in C represents 40μm. (n=3–6, * P<0.05 with respect to WT). F: The significant decrease in the expression of RyR2 at the onset of reperfusion (3min) in rat hearts was associated with a proportional decrease in Ser2815 phosphorylation such that the ratio Ser2815/RyR2 did not change significantly. [n=7–13, * P<0.05 with respect to preischemia (preisch)].

Figure 5. Mitochondria is the final effector of CaMKII-dependent cell death

Both, inhibition of mitochondrial Ca²⁺ uniporter with ruthenium red (RR) or Ru 360 (RU) and inhibition of the mPTP with cyclosporine A (CsA) diminished infarct size (A) and TUNEL positive cells (B), comparing with hearts without treatment. C. Release of cytochrome c from mitochondria to cytosol was significantly less after 45/120min IR in the presence of CaMKII inhibition with KN-93; n=3–6, *P<0.05 with respect to IR.

Figure 6. CaMKII-inhibition prevents mitochondria damage in IR

A. Significant increase in cytochrome c oxidase activity unmasked by the detergent n-Dodecyl β-D-maltoside, indicating that a higher number of intact mitochondria were isolated from hearts subjected to IR in the presence of KN-93. B. Rate constant of mitochondrial swelling calculated from the exponential fitting of light scattering records like those shown in the right panel. (n= 4–6), *P<0.05 vs. IR.