Modifications of myofilament protein phosphorylation and function in response to cardiac arrest induced in a swine model

Abstract

Cardiac arrest is a prevalent condition with a poor prognosis, attributable in part to persistent myocardial dysfunction following resuscitation. The molecular basis of this dysfunction remains unclear. We induced cardiac arrest in a porcine model of acute sudden death and assessed the impact of ischemia and reperfusion on the molecular function of isolated cardiac contractile proteins. Cardiac arrest was electrically induced, left untreated for 12 min, and followed by a resuscitation protocol. With successful resuscitations, the heart was reperfused for 2 h (IR2) and the muscle harvested. In failed resuscitations, tissue samples were taken following the failed efforts (IDNR). Actin filament velocity, using myosin isolated from IR2 or IDNR cardiac tissue, was nearly identical to myosin from the control tissue in a motility assay. However, both maximal velocity (25% faster than control) and calcium sensitivity (pCa50 6.57 ± 0.04 IDNR vs. 6.34 ± 0.07 control) were significantly (p < 0.05) enhanced using native thin filaments (actin+troponin+tropomyosin) from IDNR samples, suggesting that the enhanced velocity is mediated through an alteration in muscle regulatory proteins (troponin+tropomyosin). Mass spectrometry analysis showed that only samples from the IR2 had an increase in total phosphorylation levels of troponin (Tn) and tropomyosin (Tm), but both IR2 and IDNR samples demonstrated a significant shift from mono-phosphorylated to bis-phosphorylated forms of the inhibitory subunit of Tn (TnI) compared to control. This suggests that the shift to bis-phosphorylation of TnI is associated with the enhanced function in IDNR, but this effect may be attenuated when phosphorylation of Tm is increased in tandem, as observed for IR2. There are likely many other molecular changes induced following cardiac arrest, but to our knowledge, these data provide the first evidence that this form cardiac arrest can alter the in vitro function of the cardiac contractile proteins.

Keywords: cardiac arrest; motility; myosin; phosphorylation; resuscitation; troponin.

Figure 1

Global ischemia protocol. Ventricular fibrillation (VF) was induced as previously described (Mader et al., 2010) with a transthoracic current (100 mA at 60 Hz) and was untreated for 12 min. VF lasted for 4 min before complete electrical failure was reached (green bar) at which point circulation stopped completely (yellow bar) and finally metabolic failure is reached at ~10 min. (pink bar). Resuscitation efforts began 12 min after induction of VF, initially with manual chest compressions (MCC) and infusion of epinephrine (drug, 0.1 mg/kg). At minute 15 we attempted to restart circulation electrically (RS₁) and at 18 min. If spontaneous circulation returned (ROSC) it was maintained for 2 h and then the animals were sacrificed and cardiac muscle samples taken from the left ventricle (Ischemia + 2 h reperfusion). If ROSC was not restored then after 20 min cardiac muscle samples were taken from the LV (Ischemia + failed resuscitation). Control animals were prepared identically but did not receive the ischemia protocol (Control). ^ Indicates when a drug was administered.

Figure 2

Effect of ischemia/reperfusion on V_actin. Means ± SEM for unregulated (no Tn/Tm) actin filament velocities in the in vitro motility assay using myosin isolated from the corresponding condition. The myosin used was isolated from tissue from control, ischemia with 2 h of reperfusion (IR2) and ischemia without resuscitation (IDNR). Data were analyzed using a Kruskal–Wallis ANOVA, but all comparisons were non-significant (p > 0.05). The data represent the average actin filament velocities from 106 to 30 s videos from Control, 19 from IR2 and 8, from IDNR.

Figure 3

Representative chromatograph from myosin purification over an HIC column. The isolated myosin from porcine myocardium was additionally purified using hydrophobic interaction chromotography (HIC) based on a previously described methodology (Malmqvist et al., 2004) with minor modifications (see Methods). Chromatograph displays absorbance at 280 nm vs. elution volume. Proteins with minimal affinity for the column elute at high (1.45 M) AmSO₄ (first broad peak). After the AmSO₄ is reduced to 1.2 M myosin is released (large narrow peak). This process removed impurities including actin and likely tropomyosin, selecting for myosin and its light chains (ELC, RLC) as shown by SDS-PAGE gel (inset). In the SDS-PAGE the left lane represents the sample before HIC purification and the right lane after HIC purification.

Figure 4

Purification with HIC column enhances V_actin. Further purification of the myosin using an HIC column nearly doubled V_actin from both Control tissue and tissue exposed to ischemia (IR2) and 2 h of reperfusion (p < 0.001). However, there was no difference between conditions with either the isolated myosin or the HIC purified myosin. The data represent the average actin filament velocities from 106 to 30 s videos from Control and 19 from IR2, as well as five videos each for HIC purified Control and IR2 purified samples. ^* Indicates significance at p < 0.05.

Figure 5

Effect of ischemia/reperfusion on native thin filament velocities (V_NTF). Means ± SEM for unregulated (no Tn/Tm) actin filament velocities in the in vitro motility assay using myosin isolated from the corresponding condition. The myosin used was isolated from control, ischemia/2 h of reperfusion (IR2) and Ischemia without resuscitation (IDNR). Data analyzed using a non-parametric Kruskal–Wallis ANOVA and indicated that IDNR was significantly greater than control velocity ^*, (p < 0.05). The data represent the average actin filament velocities from 20 to 30 s videos from Control, 19 from IR2 and 16 from IDNR.

Figure 6

Velocity-pCa data. (A) Native thin filament velocity (V_NTF) plotted as a function of free [Ca⁺⁺] in –log units (pCa). Thin filaments from Control tissue are plotted with filled black dots and solid line, IR2 thin filaments with gray diamonds and solid gray line and the filaments from IDNR are plotted with dark gray boxes and a dashed gray line. Points represent mean ± SEM and the data were fit with the Hill equation (see Methods). ^* Indicates significantly (p < 0.05) different from WT V_NTF. (B) Percentage of native thin filaments moving as a function of free [Ca⁺⁺] symbols and lines same as in (A). (C) Motility index, defined as the product of V_NTF and percent moving plotted as a function of free [Ca⁺⁺]. One animal myocardium was used for each condition. The data represent the average actin filament velocities from between 3 and 20 to 30 s videos at each pCa level and for each condition.

Figure 7

LC elution profiles and data dependent MS² spectra for troponin I peptides. (A) Representative LC elution profiles for the non-phosphorylated ²³SSANYR²⁸ (red), mono-phosphorylated ²²RSSpANYR²⁸ (gray), and bis-phosphorylated ²¹RRSpSpANYR²⁸ (black) peptides. Data dependent MS² spectra for the (B) mono-phosphorylated ²²RSSpANYR²⁸ and (C) bis-phosphorylated ²¹RRSpSpANYR²⁸ peptides showing fragment ions used for peptide identification and localization of phosphate.

Figure 8

Quantification of troponin I and α-tropomyosin phosphorylation. (A) Percent phosphorylation of troponin I serines 23 and/or 24 and tropomyosin serine 283 in native thin filaments isolated from control hearts, and hearts following global ischemia that did not survive attempted resuscitation (IDNR) or survived and were subject to 2 h reperfusion (IR2). (B) Levels of mono- (serine 24) and bis-phosphorylated (serines 23 and 24) troponin I relative to the control. Three dephosphorylated control and experimental samples were prepared from each heart. ^*p < 0.01 relative to control and ^p < 0.01 relative to INDR. The difference between IR2 and IDNR was p = 0.06 for tropomyosin phosphorylation.