Role of complement C5a and histones in septic cardiomyopathy

Abstract

Polymicrobial sepsis (after cecal ligation and puncture, CLP) causes robust complement activation with release of C5a. Many adverse events develop thereafter and will be discussed in this review article. Activation of complement system results in generation of C5a which interacts with its receptors (C5aR1, C5aR2). This leads to a series of harmful events, some of which are connected to the cardiomyopathy of sepsis, resulting in defective action potentials in cardiomyocytes (CMs), activation of the NLRP3 inflammasome in CMs and the appearance of extracellular histones, likely arising from activated neutrophils which form neutrophil extracellular traps (NETs). These events are associated with activation of mitogen-activated protein kinases (MAPKs) in CMs. The ensuing release of histones results in defective action potentials in CMs and reduced levels of [Ca²⁺]i-regulatory enzymes including sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2) and Na⁺/Ca²⁺ exchanger (NCX) as well as Na⁺/K⁺-ATPase in CMs. There is also evidence that CLP causes release of IL-1β via activation of the NLRP3 inflammasome in CMs of septic hearts or in CMs incubated in vitro with C5a. Many of these events occur after in vivo or in vitro contact of CMs with histones. Together, these data emphasize the role of complement (C5a) and C5a receptors (C5aR1, C5aR2), as well as extracellular histones in events that lead to cardiac dysfunction of sepsis (septic cardiomyopathy).

Keywords: Cecal ligation and puncture (CLP); Intracellular calcium ([Ca(2+)]i); NLRP3 inflammasome; Na+/Ca(2+)exchanger (NCX); Na+/K+-ATPase; Sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA2).

Figure 1:. Quantification of C5a effects on CM Ca²⁺ homeostasis.

a. Western blotting of lysates from rat CMs indicates that C5a, in a dose-dependent manner, induced phosphorylation of phospholamban (PLB) after a one-hour period at 37°C. b. C5a application (up to 340 ng/ml) to CMs increased the intracellular Ca²⁺ amplitude (0.5 Hz pacing). c. The amplitude of spontaneous Ca²⁺ release and waves also increased with C5a dose. For frames B and C, N = 3, n = 7-27 per dose (N = number of separate experiments; n = number of CMs used in each study). d. Control frequency responses of intracellular Ca²⁺ transients. e. Frequency responses of intracellular Ca²⁺ transients following C5a exposure (45 ng/mL). f. Presence of C5a (45 ng/mL) caused elevations of intracellular Ca²⁺ as indicated by higher baseline fluorescence with increasing frequency (black circles) as opposed to no C5a (open circles). g. Relative increase (%) of basal Ca²⁺ levels after C5a addition (45 ng/mL). The greatest elevations of resting Ca²⁺ occurred at higher pacing frequencies. h. The duration of electrically paced intracellular Ca²⁺ transients was greater following C5a addition (45 ng/mL) (black bars) over a range of pacing frequencies (0.5-3Hz) as compared to absence of C5a (white bars). * p<0.05, n = 2, n = 5.

Figure 2.. C5a and Sepsis-Induced [Ca²⁺]i increases in CMs are C5a Receptor-dependent

[Ca²⁺]i in rat CMs exposed in vitro to C5a (frame a, b, and c) or after CLP (frame d and e), as determined by flow cytometry using calcium indicator fluo-3AM. a. Representative cell plots of unstimulated (using control buffer) (left line) by flow cytometry and C5a-treated (1 μg/ml, 60 min at 37°C, right line) CMs. b. [Ca²⁺]i responses of rat CMs to increasing amounts of rrC5a after 60 min at 37°C. c. Increased [Ca²⁺]i in CMs exposed in vitro to rrC5a (1 μg/ml) for the indicated periods of time. d. [Ca²⁺]i in CMs isolated from rats 8, 16, 24 and 48 hr after CLP. e. Altered levels of [Ca²⁺]i in CMs isolated after 16 hr CLP in Wt, C5aR1^−/− and C5aR2^−/− mice. For each bar, n > 8 from different CM preparations. *p <0.05.

Figure 3.. Na⁺/K⁺-ATPase staining (plasma membrane marker) on mouse cardiomyocyte (a) or on frozen heart tissue (b) from control mouse.

Green: Plasma membrane (Na⁺/K⁺-ATPase), Blue: Nuclei (DAPI)

Figure 4.. Constitutive presence of components of the NLRP3 inflammasome and their upregulation in vitro and after CLP.

In frame a. three normal mouse hearts were homogenized and lysed (TRIzol), and then subjected to RT-PCR mRNAs for GADPH, NLRP3, ASC, caspase 1 and IL-1β. As is evident, all components were detected (no reverse transcriptase, no RT). b. In vitro, mouse CMs were incubated with C5a or LPS (4 hr at 37°C). As is evident, there was upregulation o NLRP3 in CMs incubated with either agonist. c. Mouse CMs in sham mice or in CLP mice (16 hr), the latter showing a very large increase (>50 fold) in mRNA for IL-1β. For each bar, n = 5. d. Frozen ventricles 12 hr after CLP stained for IL-1β.

Figure 5.. Proposed cascade of events after CLP leading to “cardiomyopathy of sepsis”

Briefly, complement C5a and extracellular histones induce reductions in key Ca²⁺ regulatory proteins as well as Na⁺/K⁺-ATPase protein in CMs leading to inability to clear cytosolic Ca²⁺, together with activation NLRP3 inflammasome and MAPKs resulting in the cardiomyopathy of sepsis.