CaMKII-dependent ryanodine receptor phosphorylation mediates sepsis-induced cardiomyocyte apoptosis

Abstract

Sepsis is associated with cardiac dysfunction, which is at least in part due to cardiomyocyte apoptosis. However, the underlying mechanisms are far from being understood. Using the colon ascendens stent peritonitis mouse model of sepsis (CASP), we examined the subcellular mechanisms that mediate sepsis-induced apoptosis. Wild-type (WT) CASP mice hearts showed an increase in apoptosis respect to WT-Sham. CASP transgenic mice expressing a CaMKII inhibitory peptide (AC3-I) were protected against sepsis-induced apoptosis. Dantrolene, used to reduce ryanodine receptor (RyR) diastolic sarcoplasmic reticulum (SR) Ca²⁺ release, prevented apoptosis in WT-CASP. To examine whether CaMKII-dependent RyR2 phosphorylation mediates diastolic Ca²⁺ release and apoptosis in sepsis, we evaluated apoptosis in mutant mice hearts that have the CaMKII phosphorylation site of RyR2 (Serine 2814) mutated to Alanine (S2814A). S2814A CASP mice did not show increased apoptosis. Consistent with RyR2 phosphorylation-dependent enhancement in diastolic SR Ca²⁺ release leading to mitochondrial Ca²⁺ overload, mitochondrial Ca²⁺ retention capacity was reduced in mitochondria isolated from WT-CASP compared to Sham and this reduction was absent in mitochondria from CASP S2814A or dantrolene-treated mice. We conclude that in sepsis, CaMKII-dependent RyR2 phosphorylation results in diastolic Ca²⁺ release from SR which leads to mitochondrial Ca²⁺ overload and apoptosis.

Keywords: CaMKII; Sepsis; apoptosis; mitochondrial dysfunction; ryanodine receptors.

Figure 1

Polymicrobial model of sepsis is associated with apoptosis. Evaluation of activation of the apoptotic cascade. Representative blots and overall results showing that hearts of WT‐CASP mice have a significant increase the ratio between Bax and Bcl‐2 (Bax/Bcl2) relative to Sham controls (n = 4). WT‐CASP exhibited a significant decrease in the mitochondrial expression of cytochrome C (CytoC) compared to WT‐Sham mice (n = 4) (unpaired t test). Myocardial mitochondrial inner membrane potential (ΔΨm) changes were examined using rhodamine‐123 (RH‐123) fluorescence. As shown, ΔΨm measured in isolated mitochondria was significantly lower in WT‐CASP compared with WT‐Sham mice (n = 4) (Mann‐Whitney test). The bottom panel shows typical images and overall results of the increase in TUNEL‐positive cells in myocytes isolated from CASP mice compared to Sham myocytes. Scatter plot shows values from 3‐4 independent myocyte isolations (3‐4 hearts) P < .05 (unpaired t test). Data are expressed as means ± SEM * P < .05 vs Sham

Figure 2

CaMKII mediates sepsis‐induced cardiac apoptosis. Representative immunoblots and overall results from heart homogenates and isolated mitochondria from CASP‐ and Sham‐operated transgenic mice expressing a CaMKII inhibitory peptide (AC3I) or mice expressing the scrambled control peptide (AC3C). CASP‐AC3C mice show a significant increase in Bax/Bcl2, a significant decrease in mitochondrial cytochrome C expression (unpaired t test) and a significant decrease in ∆ψm compared to Sham AC3C (n = 4) (Mann‐Whitney test). AC3I‐CASP mice did not show altered apoptotic indexes compared to mice to AC3I‐Sham mice (n = 4) indicating that CaMKII inhibition protects hearts from sepsis‐induced‐apoptosis. Data are expressed as means ± SEM * P < .05 vs Sham

Figure 3

Preventing RyR2 phosphorylation protects against apoptosis induced by sepsis in mice. Typical Western blots, images and overall results showing that CASP‐operated S2814A mice do not show significant alterations in the apoptotic indexes Bax/Bcl2, mitochondrial cytochrome C expression, ∆ψm or TUNEL staining (n = 4 per group) compared to Sham‐operated S2814A mice. All data are expressed as means ± SEM * P < .05 (unpaired t test) vs Sham)

Figure 4

Reducing RyR2 open probability prevents sepsis‐induced‐apoptosis. Typical blots and overall results of the effect of dantrolene pretreatment on sepsis‐induced apoptosis. WT‐CASP + dantrolene mice hearts showed non‐significant (NS) changes in Bax/Bcl2, mitochondrial cytochrome C expression and ∆ψm compared to Sham + dantrolene‐operated WT mice (n = 4 per group). All data are expressed as means ± SEM * P < .05 (unpaired t test) vs Sham)

Figure 5

Role of mitochondrial Ca²⁺ in sepsis. A, Typical tracings of changes in the fluorescence of Ca²⁺ indicator Calcium Green‐5N. Arrows indicate Ca²⁺ addition to the mitochondrial suspension. Increasing extramitochondrial Ca²⁺ results in a rapid increase in the fluorescent signal followed by a decline in the fluorescence intensity of the sensor resulting from mitochondrial Ca²⁺ uptake. WT‐Sham mitochondria showed enhanced tolerance to extramitochondrial Ca²⁺ addition compared to WT‐CASP mitochondria. The measurement of extramitochondrial Ca²⁺ shows that WT‐Sham cardiac mitochondria are able to accumulate significantly more Ca²⁺ than WT‐CASP mitochondria (CRC) (n = 4). Mann‐Whitney test results are expressed as mean ± SEM. B: Typical traces and overall results of changes of Calcium Green fluorescence after Ca²⁺ addition in samples of mitochondria from S2814A mutant CASP mice CASP mice compared to Sham. Mitochondria from CASP mice showed preserved CRC compared to Sham (n = 4). Unpaired t test, results are expressed as mean ± SEM