Article, Resuscitation

Cardiopulmonary resuscitation ameliorates myocardial mitochondrial dysfunction in a cardiac arrest rat model

a b s t r a c t

Purpose: Previous studies implicate that the mitochondrial injury may play an important role in the development of post-resuscitation myocardial dysfunction, however few of them are available regarding the ultrastructural alterations of myocardial mitochondria, mitochondrial energy producing and utiliza- tion ability in the stage of arrest time (no-low) and resuscitation time (low-flow). This study aimed to observe the dynamic changes of myocardial mitochondrial function and Metabolic disorders during car- diac arrest (CA) and following cardiopulmonary resuscitation (CPR).

Methods: A total of 30 healthy male Sprague-Dawley rats were randomized into three groups: 1) VF/CPR: Ventricular fibrillation (VF) was electrically induced, and 5 min of CPR was performed after 10 min of untreated VF; 2) Untreated VF: VF was induced and untreated for 15 min; and 3) Sham: Rats were iden- tically prepared without VF/CPR. Amplitude Spectrum Area at VF 5, 10 and 15 min were calcu- lated from ECG signals. The rats’ hearts were quickly removed at the predetermined time of 15 min after beginning the procedure to gather measurements of myocardial mitochondrial function, high- energy phosphate stores, lactate, mitochondrial ultrastructure, and myocardial glycogen.

Results: The mitochondrial respiratory control ratios significantly decreased after CA compared to Sham group. CPR significantly increased respiratory control ratios compared with untreated VF animals. A sig- nificant decrease of myocardial glycogen was observed after CA, and a more rapid depletion of myocar- dial glycogen was observed in CPR animals. CPR significantly reduced the tissue lactate. The mitochondrial ultrastructure abnormalities in CPR animals were less severe than untreated VF animals. AMSA decayed during untreated VF; however, it was significantly greater in CPR group than the untreated VF group. In addition, AMSA was clearly positively correlated with ATP, but negatively corre- lated with myocardial glycogen.

Conclusion: Impairment of myocardial mitochondrial function and the incapability of utilizing glycogen were observed after CA. Furthermore, optimal CPR might, in part, preserved mitochondrial function and enhanced utilization of myocardial glycogen.

(C) 2019

  1. Introduction

Cardiac arrest with cardiopulmonary resuscitation (CPR) is an event of global myocardial ischemia/reperfusion. Post- resuscitation myocardial dysfunction has been observed after the

* Corresponding author at: Department of Emergency Medicine, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, 107 Yan Jiang Xi Road, Guangzhou, Guangdong 510120, China.

E-mail address: [email protected] (X. Fang).

1 These authors contributed equally to this study.

return of spontaneous circulation (ROSC) in both animal [1,2] and human studies [3], and remains the leading cause of early death after ROSC [4].

Mitochondria are pivotal intermediates for myocardial ische- mia/reperfusion injury. It is widely acknowledged that ischemia/ reperfusion leads to mitochondrial ultrastructural and functional injury, demonstrated in models ranging from the intact heart to structural and functional characterizations of individual molecular components of mitochondria [5-7]. In a series of experiments using small and big animal models receiving ventricular fibrillation (VF)/ 0735-6757/(C) 2019

CPR, Gazmuri et al. demonstrated that post-resuscitation myocar- dial dysfunction is correlated with mitochondrial injuries [8-11]. We recently have found the impairment of energy-production and ultrastructural abnormalities of myocardial mitochondria (exhibited as irregular shape, diffuse distribution, mitochondrial swelling and edema, outer-membrane rupture and loss of inner- membrane cristae with amorphous densities), as evidenced by the body’s incapability to utilize energy substrates during VF/CPR and the development of post-resuscitation myocardial dysfunction in a rat model of VF/CPR [12,13]. The work summarized above implicates that mitochondrial-associated metabolic disorders dur- ing CA and after successful CPR might contribute to post- resuscitation myocardial dysfunction.

Though there are series of experiments to implicate that the mitochondrial injury may play an important role in the develop- ment of post-resuscitation myocardial dysfunction, yet at present, limited data are available to regard the changes in myocardial oxidative phosphorylation, including how they utilize energy sub- strates and their capacity to produce high-energy phosphate dur- ing CA and following CPR, representing the two periods of ”no- flow” and ”low-flow,” respectively. The current study aims to investigate the dynamic changes of mitochondrial function, their energy-producing ability, lactate accumulation, and capability to utilize energy substrates (typically glycogen) during CA and fol- lowing CPR.

In the 2015 American Heart Association Guideline for Car- diopulmonary Resuscitation, CPR is the primary rescue measure for cardiac arrest patients. We hypothesized that CA provokes sig- nificant impairment of myocardial mitochondrial function, decreases high energy phosphate compounds, causes accumula- tion of tissue lactate, as well as renders incapable utilization of tis- sue glycogen. We further hypothesized that even ”low blood perfusion” generated by CPR can ameliorate the metabolic disorder of anaerobic glycolysis and oxidative phosphorylation via preserv- ing mitochondrial function. This would explain the cardioprotec- tive effects of CPR from the specific molecular mechanism.

  1. Methods

The present protocol was approved by the Animal Care and Use Committee of Sun Yat-sen University. All animals were cared for humanely and in compliance with the ”Principles of Laboratory Animal Care” formulated by the Ministry of Science and Technol- ogy of the People’s Republic of China.

Animal preparation

A total of 30 healthy male Sprague-Dawley rats, aged 6- 8 months and weighing between 450 and 550 g, were supplied by the Experimental Animal Center of Traditional Chinese Medi- cine University of Guangzhou. All animals were fasted overnight but had free access to water. After inhaling CO2 for brief anesthesia, animals were anesthetized by an intraperitoneal injection of pen- tobarbital (45 mg/kg); additional doses (10 mg/kg) were adminis- tered at 1-h intervals or when needed to maintain anesthesia. A PE-50 catheter was advanced through the left femoral artery into the aorta to measure the mean aortic pressure (MAP). After animal preparation and recording baseline information, all animals were randomly assigned to three groups using sealed envelope. For induction of VF, a 4 French PE catheter was advanced through the right external jugular vein into the right atrium, and through its lumen a pre-curved guide wire was advanced into the right ven- tricle for inducing VF. Rectal core temperature was monitored and body temperature was maintained through a heat lamp at

36.8 +- 0.2 ?C. Hemodynamic data and ECG were continuously

recorded via a WinDaq data-acquisition system (DataQ, Akron, Ohio, USA). Before onset of VF, 0.5 ml of blood was withdrawn from the arterial catheter to measure the arterial blood gas using an i-STAT portable analyzer (Abbot, Abbott Park, IL, USA).

Experimental procedure

Animals were randomized into three groups: 1) VF/CPR group (n = 10): VF was electrically induced and untreated for 10 min, fol- lowing by 5 min of CPR; 2) Untreated VF group (n = 10): VF was induced and maintained for 15 min without CPR attempt; and 3) Sham control (n = 10): animals were submitted to identical anes- thetic and surgical procedures without VF/CPR.

Ten minutes prior to inducing VF, the animals were mechani- cally ventilated with a tidal volume of 0.55 mL/100 g of body weight, a frequency of 100 breaths/min and inspired O2 fraction (FiO2) of 0.21. VF was electrically induced with a progressive increase in 60-Hz current to a maximum of 3 mA delivered to the right ventricular endocardium. To prevent spontaneous rever- sal of VF, the current flow was continued for 3 min. Mechanical ventilation was discontinued during VF. For the CPR group, contin- uous precordial compression and mechanical ventilation with 100% O2 were applied for 5 min after 10 min untreated VF. Precor- dial compression at a rate of 250 min–1 was synchronized to pro- vide a compression/ventilation ratio of 2:1. Compression depth was adjusted to maintain diastolic aortic pressure at 27 +- 2 mmHg [14]. VF was untreated for 15 min without CPR for the VF-only group. All animals were immediately sacrificed after 15 min of VF with or without CPR. The heart of each animal was rapidly resected. The apex was removed (an approximately 150 mg piece) and flash of apex frozen in liquid nitrogen for further analysis of myocardial high-energy phosphate stores and lactate by high per- formance liquid chromatography (HPLC). The left ventricles were quickly separated, and then serially cut into smaller pieces for the measurements of mitochondrial Respiratory function, the mor- phological examination of altered mitochondria ultrastructure, and myocardial glycogen.

Quantitative electrocardiographic waveform measure

The ECG signal was collected from a standard lead II configura- tion of surface electrodes. Analog ECG signals were digitized and converted from a time to a frequency domain by fast Fourier trans- formation. Amplitude spectrum area (AMSA) at VF 5, 10 and 15 min were calculated as the sum of the products of individual fre- quencies between 4 and 48 Hz. The AMSA value of each 4-s time segment also was calculated according to the equation: AMSA = R Ai . fi. All analyses were carried out in the MATLAB 2014a Signal Processing Toolbox.

Isolation of myocardial mitochondria

The mitochondria were isolated from free left ventricular (LV) walls using a Tissue Mitochondria Isolation Kit (C3606, Beyotime Institute of Biotechnology, Shanghai, China) following manufac- turer’s directions as follows. A LV piece approximately 150- 200 mg was quickly sampled and placed in ice-cold PBS. The ice- cold trypsin (from the Kit) was discarded after 20 min incubation of small pieces sample. The tissue was placed in an ice-cold Dounce homogenizer (Wheaton, Millville, NJ) and the Mitochondrial Extraction Buffer (supplied by the kit) was added. The homoge- nates were centrifuged (600g, 4 ?C 5 min) after grinding 25 times (15 with a loosely-fitting pestle and 10 with a tightly-fitting pestle) to ensure that no chunks of tissue remained. To settle mitochon- dria, the supernatant was collected for a second centrifuge (11,000g,4 ?C 10 min). Finally, the mitochondria were resuspended

and preserved in a Storage Buffer (supplied by the kit). The suspen- sion of mitochondria was then stored in an ice bath before testing. The protein concentration of each sample was measured by a Qubit fluorometer (Invitrogen, Carlsbad, CA). All above operations were performed in a 0-4 ?C ice bath.

Ultrastructural examination in mitochondria by transmission electron microscope (TEM)

Tissue blocks were obtained from subendocardial myocardium of the left ventricle. Samples were fixed in cold 2.5% glutaralde- hyde with 0.1 mol/L cacodylate buffer (pH 7.4), post-fixed in 1% osmium tetroxide, dehydrated, and embedded in Epon. According to the stereological principle of randomization, ultrathin sections (60-80 nm) were randomly cut regardless of the orientation and mounted on copper grids, stained with lead citrate and uranyl acet- ate, and then viewed with a FEI Tecnai G2 transmission electron microscope equipped with a Gatan 832 CCD camera (Gatan, Pleasanton, USA) at a final magnification of x13,500.

Mitochondrial respiration function

The respiration rates of mitochondria (state 3, ADP-stimulated and state 4, ADP-deleted) was analyzed by a Clark oxygen elec- trode (Hansatech Instruments, Norfolk, U.K.) in a chamber which was sealed, thermostatically controlled to 25 ?C, and continuously stirred. The experiments were conducted using a commercially available kit (GMS10097, GENMED, Shanghai, China) to measure the mitochondrial respiration rates, according to the manufac- turer’s instructions, as follows. Reaction buffer (reagent A) was used for incubation of mitochondrial fractions. Reagent B for state 4 substrate solution and reagent C for state 3 substrate solution were added in sequence. State 3 and state 4 respiration rates of mitochondria were determined. The unit of mitochondrial respira- tion rate was oxygen consumption per nM/min/mg protein (expressed in nmoL oxygen/min/mg mitochondrial protein); and mitochondrial respiratory control ratios (RCR) was calculated as RCR = state 3 respiration rate/state 4 respiration rate. RCR presents the oxidative phosphorylation and integrity of the membrane in the mitochondrion. Meanwhile, the reduction in RCR indicates impairment of mitochondrial function.

Glycogen content

The glycogen contents were determined using freshly removed heart tissue samples by the anthrone reagent method via a glyco- gen assay kit (Nanjing Jiancheng Bioengineering Institute, China) following the manufacturer’s instructions. This method is based on a colorimetric product formed when polysaccharide is dissolved in water and hydrolyzed with sulfUric acid containing the anthrone reagent. The solution is then allowed to cool, and the absorbance of the resulting solution, and glucose as standard, was measured at 620 nm. The result was expressed as milligrams of glycogen per gram of myocardial tissue (mg/g of tissue), as per the manufac- turer’s instructions.

Determination of myocardial high-energy phosphate stores and lactate by HPLC

Frozen samples were weighed then homogenized (150 mg of tissue/ml of acid) in 0.3 M perchloric acid. The homogenates were centrifuged at 4 ?C for 5 min at 3000 rpm before supernatants were collected for analyses. The supernatants were adjusted to pH 7.6-

7.8 with 0.5 M KOH. The extracts were centrifuged again at 4 ?C for 5 min at 3000 rpm, then stored in liquid nitrogen before testing via HPLC using an Agilent Technologies 1200 Series (Germany)

with a diode array detector. Reversed-phase HPLC was performed on an Inertsil ODS-3 column (4.6 x 250 mm i.d.; 5 lm, GL Science) for measurements of lactate and high-energy phosphate stores, including phosphocreatine (PCr), adenosine triphosphate (ATP). The mobile phase buffer was composed of 0.2 M potassium dihy- drogen phosphate/dipotassium hydrogen phosphate and 3 mM tetrabutylammonium chloride at a pH of 6.8. The mobile phase components, buffer (pH 6.8): methanol (80:20 v/v), were then mixed and finally filtered through membrane filters of 0.2 lm. The mobile phase was pumped at a flow rate of 0.8 mL/min and the eluents were monitored at a wavelength of 260 nm for effec- tive separation and resolution of the analyte peaks.

Statistical analyses

The outcomes (including state3, state 4, PCr, ATP, Lac, Glycogen content) in our study were continuous data. Normality of these data was confirmed by the test of normal distribution and homo- geneity of variance. All statistical analyses were performed using SPSS version 17.0 (SPSS, Chicago, IL). After confirmation of normal distribution with the Kolmogorov-Smirnov-Test, all variables above were compared either using parametric tests (ANOVA) or non-parametric tests (Mann-Whitney-U-test). For the measure- ments among these groups, ANOVA followed by Scheffe multiple- comparison techniques were employed.

For the analysis of AMSA data, ANOVA repeated measurement testing was used. Linear correlations were calculated using the Pearson correlation coefficient. All Measurements are presented as means +- standard deviation (SD). A P value of <0.05 was consid- ered as statistically significant.

  1. Results
    1. Baseline physiologies

Baseline physiologies of animals in all groups are illustrated in Table 1. There were no differences among these groups.

Mitochondrial respiration function

To investigate the alterations in mitochondrial function, we employed oxygen electrode experiments to evaluate mitochondrial respiration. As shown in Fig. 1, the ADP-stimulated state 3 activi- ties were significantly reduced in animals suffering from prolonged cardiac arrest either with or without CPR attempts, compared with sham control animals (P < .05). However, the state 3 respiration in VF/CPR animals was significantly improved compared to those in the untreated VF animals group (P < .05). There were no significant differences in non-ADP-stimulated state 4 activities among the groups.

RCR is a key parameter of the mitochondrial function and indi- cates the redox state of the system. Animals undergoing 15 min of either treated or untreated VF had significantly decreased RCR

Table 1

Baseline information.

VF/CPR group

VF group

Control group

Weight, g

489 +- 25

503 +- 20

495 +- 15

Heart rate, beats/min

383 +- 15

389 +- 19

400 +- 16

Mean artery pressure, mm Hg

129 +- 8

122 +- 9

133 +- 9


7.46 +- 0.03

7.49 +- 0.02

7.47 +- 0.03

ETCO2, mm Hg

35+- 4

39 +- 6

38+- 5

Lactate, mmol/L

0.58 +- 0.06

0.59 +- 0.05

0.65 +- 0.03

Values are presented as mean +- SD. VF, ventricular fibrillation; CPR, cardiopul- monary resuscitation.

ultrastructural changes of mitochond”>Fig. 1. The mitochondrial respiration ability. VF, ventricular fibrillation; CPR, cardiopulmonary resuscitation. *P < .05 vs. the Control group; **P < .01 vs. the Control group;

#P < .05 vs. the VF/CPR group.

levels compared with sham control animals. Five minutes of effec- tive CPR, including chest compression and mechanical ventilation, partially restored the mitochondrial RCR compared with the untreated VF animals.

Myocardial high-energy phosphate stores and lactate

The content of PCr and ATP in both VF animals and VF/CPR ani- mals were less than those in the control animals (P < .05). Decreases of high-energy phosphate stores were observed in CA animals when compared with the sham animals. Yet, no differ- ences were observed for the decreased high-energy phosphate stores between VF/CPR animals and Untreated VF. Meanwhile, sig- nificant increases of the concentration in myocardial lactate were observed in both VF/CPR animals and untreated VF animals (P < .05), of which the myocardial lactate concentration in VF/CPR animals was less than that of untreated VF animals (P < .01) (Fig. 2).

Myocardial glycogen contents

The myocardial glycogen contents in both VF/CPR animals and untreated VF animals were significantly less than those of control animals (P < .01). Moreover, we observed that glycogen levels in VF/CPR animals were obviously decreased in comparison with untreated VF animals (P < .01) (Fig. 3).

Quantitative electrocardiographic waveform measure

AMSA values significantly decreased during untreated VF in both VF and VF/CPR animals. However, much greater AMSA during CPR was achieved by the VF/CPR group in comparison with the VF group. In addition, there was a marked and positive relationship between AMSA at VF 15 min and ATP; but myocardial glycogen content was significantly and negatively correlated with AMSA at VF 15 min (Fig. 4).

ultrastructural changes of mitochondria

The representative photographs of each group are shown in Fig. 5. The mitochondria in sham control animals were irregular in shape and were compacted with a higher electron-dense matrix. In contrast, the mitochondria in the untreated VF animals showed significant expansion with reduced matrix density. In the VF/CPR animals group, the mitochondria were slightly swollen, with a rel- atively normal matrix density. Moreover, intramyocellular lipid (IMCL) droplets are only detected in VF/CPR animals. Overall, the mitochondrial ultrastructure of VF/CPR animals displayed Minor injury as compared with sham animals but was less severe than that of VF animals.

  1. Discussions

It is acknowledged that low flow reperfusion generated by CPR. Therefore, pathophysiological impairment after ischemia/reperfu- sion from cardiac arrest and successful resuscitation is determined by three episodes, including arrest time (no-flow), resuscitation time (low-flow), and the reperfusion phase following ROSC. The pathological features of this model are therefore distinguishable from other complete ischemia/reperfusion settings. Several studies on its mechanisms have failed to ascertain distinct intrinsic patho- logical features [15,16]. To date, there are abundant evidences sug- gested that the Quality of CPR during CA is associated with post- resuscitation outcomes [17,18]. As well as good CPR, Therapeutic hypothermia has been reported to attenuate global cerebral reperfusion-induced mitochondrial damage and mitochondria- mediated apoptosis. The ultrastructural changes underlying its Neuroprotective effects of TH had been reported. However, the pathophysiological changes during CA and CPR are much less fre- quently investigated, which may bear logical relationships to the mechanism of post-resuscitation myocardial dysfunction.

Ischemia and reperfusion lead to mitochondrial, as well as cel- lular damage in cardiac cells [5-7]. Recently, studies have sug- gested Mitochondrial dysfunction as one of the key factors in the

Fig. 2. Myocardial high-energy phosphate stores and lactate. PCr, phosphocreatine; ATP, adenosine triphosphate; Lac, lactate; VF, ventricular fibrillation; CPR, cardiopulmonary resuscitation. *P < .05 vs. the Control group; **P < .01 vs. the Control group; #P < .05 vs. the VF/CPR group.

Fig. 3. Myocardial glycogen contents. VF, ventricular fibrillation; CPR, cardiopul- monary resuscitation. *P < .05 vs. the Control group; **P < .01 vs. the Control group; #P < .05 vs. the VF/CPR group.

development of post-resuscitation myocardial dysfunction [19,20]. Studies also indicate that targeting metabolic changes and redox imbalance during CA and resuscitation may provide a promising

strategy to ameliorate post-resuscitation myocardial injury [19- 23]. Previously, Yeh et al. suggested that in spite of low perfusion and oxygen delivery, CPR is able to preserve myocardial mitochon- drial function, mainly by the preservation of Complex I activity during ischemia [24]. More recently, in a model of CA and resusci- tation, our group identified the presence of ultrastructural abnor- malities in mitochondria, further evidenced by the body’s inability to utilize energy substrates and the impaired energy pro- duction during CA and following successful resuscitation [12]. The effort to minimize post resuscitation myocardial injury prompted current research further into dynamic changes of myocardial mito- chondrial function and Energy metabolism during the no- or low- flow intervals of CA and resuscitation.

In the present study, impaired cardiac mitochondrial function as measured by RCR was observed after CA. This reduction in RCR was due to reduced state 3 respiration and unchanged state 4 respiration. However, animals that received CPR had a lesser reduction of RCR when compared with untreated VF animals. Con- currently, a reduction in mitochondrial respiration may be related to abruptly decrease myocardial contents of PCr and ATP, signifi- cant increases of tissue lactate level, and a marked decrease of AMSA following cardiac arrest. When optimal CPR was performed, accumulation of myocardial lactate and the values of AMSA were

Fig. 4. Changes in the AMSA during VF (A) and the relationships between AMSA and ATP (B) or myocardial glycogen contents (C). AMSA, Amplitude spectrum area; ATP, adenosine triphosphate; VF, ventricular fibrillation; CPR, cardiopulmonary resuscitation. #P < .05 vs. the VF/CPR group.

partially recovered. In parallel with the better preservation of mitochondrial function, the more effective utilization of myocar- dial glycogen was observed in the CPR animals when compared with the untreated VF animals.

Mitochondrial RCR is a key parameter of mitochondrial func- tion and indicates the redox state of the system. In this study, the comparisons of RCR levels among groups suggest that RCR levels are significantly depressed during both the no-flow periods of untreated VF and CPR. standard CPR by itself is inherently inefficient, providing only 10%-30% of normal perfusion to the heart even when delivered according to guidelines [25,26]. In this study, RCR levels were significantly improved after standard CPR, although they still remained significantly lower than control ani- mals’ levels. Our findings suggested that even a low flow gener- ated by resuscitation efforts may ameliorate mitochondrial dysfunction during cardiac arrest. Our observation is consistent with the studies by Yeh et al. who reported 10 min of CPR fol- lowing an untreated 15 min CA maintains RCR at the level seen with 15 min CA alone in a rat model of prolonged CA [24]. In our current study, the preservation of mitochondrial respiratory function following CPR is further supported by the finding of mild ultrastructural alterations of mitochondria in CPR animals, contrasting to the more severe morphological alterations in VF animals.

Consistent with the trend of mitochondrial respiration ineffi- ciency and morphological alteration, mitochondrial energy- producing ability, as measured by the contents of myocardial PCr Declaration of interest statement”>and ATP, was better preserved in CPR animals than those in untreated VF animals. In the aerobic setting, virtually all (~95%) ATP generated in the heart arises from mitochondrial oxidative phosphorylation [27,28]. Fatty acid and carbohydrate oxidation ceases during global myocardial ischemia following CA. Myocardial function is now dependent on anaerobic glycolysis. Only about 5% of the energy normally produced by oxidation of glucose is pro- vided by this emergency pathway. Therefore, the requirement of energy in the fibrillating heart rapidly exceed availability. The residual ATP is hydrolyzed, and anaerobically generated lactate accounts for excesses of protons, specifically H+ [29]. Thus, a signif- icant accumulation of myocardial lactic acid was observed after CA either with or without CPR in this study. The reduction of tissue lactate in the CPR animals indicated that although inadequate myocardial perfusion and oxygen delivery are generated by CPR, it still partially drives oxidative phosphorylation and help reduce anaerobic glycolysis.

Significant decreases in myocardial glycogen contents were

observed after CA in our current investigation. We further observed more rapid depletion of myocardial glycogen in CPR ani- mals compared with the untreated VF animals. This is significant because when CA occurs, there is a combined effect of anoxia and lack of substrate. The oxidative phosphorylation reaction soon stops due to depleted oxygen supply. Despite its low efficiency resulting in intracellular acidosis, the tissues use stored glycogen for anaerobic glycolysis in an attempt to maintain tissue energy supply. This may explain the decrease of myocardial glycogen con- tents after CA. Although the oxygen delivery to the heart during CPR is limited, the less impaired mitochondrial function may more effectively utilize the stored myocardial glycogen for oxidative phosphorylation.

In the present study, the results of transmission electron micro- graphs have shown IMCL droplets accumulations are present in VF/ CPR animals. Accumulations of IMCL droplets might be sign of the incapability of mitochondria in utilizing energy substrates. Fat dro- plets indicate that blood flow enters the myocardium during CPR. However, the myocardium cannot use lipid droplets at this time. It can only use glycogen, indicating indirectly that effective CPR can provide limited perfusion to the heart.

In the VF phase, the heart’s energy will gradually decrease until the cardiomyocytes are completely dead if no CPR is applied, and the depletion of myocardial energy during VF could be reflected from ECG signals [30,31]. In the present study, the AMSA value during the untreated VF phase was decreased in a time dependent manner, and it increased as CPR was applied. Consistent with our findings, previous research also showed that AMSA is increased by effective chest compressions and its value was associated with the concentration of ATP [31,32]. Moreover, we also found myocar- dial glycogen content is negatively relative to AMSA during VF and CPR. Although the low perfusion to the heart during CPR is limited, the less impaired mitochondrial function may more effectively uti- lize the stored myocardial glycogen for oxidative phosphorylation, resulting in a much greater AMSA achieved by CPR. Therefore, the AMSA which reflects the myocardial activity might also serve as a quality-control for CPR, revealing whether the Myocardial blood flow was improved.

We admit several limitations in our findings. (1) In the aerobic setting, fatty acid b-oxidation accounts for the majority of oxida- tive energy metabolism (60-80%), while carbohydrate metabolism accounts for the remaining 20-40% [33]. In the current investiga- tion, we did not measure the intramyocellular lipid content. Con- sidering the fact that anaerobic glycolysis predominates during CA and CPR, we elected to report the glycogen content only, for the purpose of generally outlining the metabolic disorder under the aerobic and anaerobic conditions of CA and resuscitation. (2) the current study is designed to characterize the mitochondrial- associated metabolic disorders during CA and CPR. The mitochon- drial dysfunction after successful resuscitation deserves further investigation. (3) In this study, all animals were randomly assigned to the three groups using sealed opaque envelope However, we could not fully guarantee that the process of VF and CPR was com- pletely blinded. This is added as a study limitation. The mitochon- drial functional and structural testing were performed by technicians who were blinded to the grouping.

  1. Conclusions

In this rat model of CA and CPR, the presence of metabolic dis- orders, significant impairment of myocardial mitochondrial func- tion associated with metabolic disorders and the incapability to utilize glycogen were observed after CA. We further observed that even low blood perfusion generated by standard CPR might pre- serve mitochondrial function and enhancing utilization of stored myocardial glycogen.

Declaration of interest statement



Fig. 5. Representative transmission electron micrographs (magnification 13,500x). (A) Control; The mitochondria are irregular in shape, and compact with a higher electrondense matrix. No intramyocellular lipid (IMCL) droplets are detected. (B) VF; The mitochondria are prominently swollen with reduced matrix density. No IMCL droplets are detected. (C) VF + CPR. The majority of mitochondria have moderately swelling with reduced matrix density. IMCL droplets accumulations are present.Control: Animal was submitted to identical anesthetic and surgical procedures without ventricular fibrillation (VF) and cardiopulmonary resuscitation (CPR); VF: Animal was electrically induced and maintained VF for 15 min without CPR attempt; VF + CPR: Animal was electrically induced, and 5 min of CPR was performed followed by 10 min of untreated VF.

Funding statement

This study was supported by the grants from the National Nat- ural Science Foundation of China (81272061), the project of Lead- ing Talents in Pearl River Talent Plan of Guangdong Province (No. 81000-42020004) and the project of Guangdong Science and Tech- nology Department (No. 2013B021800038).


  1. Babini G, Grassi L, Russo I, Novelli D, Boccardo A, Luciani A, et al. Duration of untreated cardiac arrest and clinical relevance of Animal experiments: the relationship between the ”No-Flow” duration and the severity of post-cardiac arrest syndrome in a porcine model. Shock 2018:49.
  2. Wang W, Hua T, Li H, Wu X, Bradley J, Peberdy MA, et al. Decreased cAMP level and decreased downregulation of b1-adrenoceptor expression in therapeutic hypothermia-resuscitated myocardium are associated with improved post- resuscitation myocardial function. Journal of the American Heart Association Cardiovascular & Cerebrovascular Disease 2018;7(6).
  3. Cha KC, Kim HI, Kim OH, Yong SC, Kim H, Kang HL, et al. Echocardiographic patterns of postresuscitation myocardial dysfunction. Resuscitation 2018;124:90.
  4. JP N, J S, A C, T C, VR M, CD D, et al. Erratum to: European Resuscitation Council and European Society of Intensive Care Medicine 2015 guidelines for post- resuscitation care. Intensive Care Med 2015;41(12):2039-56.
  5. Solaini G, Harris DA. Biochemical dysfunction in heart mitochondria exposed to ischaemia and reperfusion. Biochem J 2005;390(Pt 2):377-94.
  6. Lesnefsky EJ, Moghaddas S, Tandler B, Kerner J, Hoppel CL. Mitochondrial dysfunction in cardiac disease: ischemia-reperfusion, aging, and heart failure. J Mol Cell Cardiol 2001;33(6):1065-89.
  7. Schwarz K, Siddiqi N, Singh S, Neil CJ, Dawson DK, Frenneaux MP. The breathing heart – mitochondrial Respiratory chain dysfunction in cardiac disease. Int J Cardiol 2014;171(2):134-43.
  8. Ayoub IM, Kolarova JD, Kantola RL, Radhakrishnan J, Wang S, Gazmuri RJ. Zoniporide preserves left ventricular compliance during ventricular fibrillation and minimizes postresuscitation myocardial dysfunction through benefits on energy metabolism. Crit Care Med 2007;35(10):2329-36.
  9. Ayoub IM, Radhakrishnan J, Gazmuri RJ. Targeting mitochondria for resuscitation from cardiac arrest. Crit Care Med 2008;36(11 Suppl):S440.
  10. Huang CH, Tsai MS, Chiang CY, Su YJ, Wang TD, Chang WT, et al. Activation of mitochondrial STAT-3 and reduced mitochondria damage during hypothermia treatment for post-cardiac arrest myocardial dysfunction. Basic Res Cardiol 2015;110(6):59.
  11. Radhakrishnan J, Wang S, Ayoub IM, Kolarova JD, Levine RF, Gazmuri RJ. Circulating levels of cytochrome c after resuscitation from cardiac arrest: a marker of mitochondrial injury and predictor of survival. Am J Physiol Heart Circ Physiol 2007;292(2):H767.
  12. Fang X, Huang Z, Zhu J, Jiang L, Li H, Fu Y, et al. Ultrastructural evidence of mitochondrial abnormalities in postresuscitation myocardial dysfunction. Resuscitation 2012;83(3):386.
  13. Tsai MS, Huang CH, Tsai SH, Tsai CY, Chen HW, Cheng HJ, et al. The difference in myocardial injuries and mitochondrial damages between asphyxial and ventricular fibrillation cardiac arrests. Am J Emerg Med 2012;30(8):1540-8.
  14. Fang X, Huang L, Sun S, Weil MH, Tang W. Outcome of prolonged ventricular fibrillation and CPR in a rat model of chronic ischemic left ventricular dysfunction. Biomed Res Int 2013;2013:564501.
  15. Song F, Shan Y, Cappello F, Rappa F, Ristagno G, Yu T, et al. Apoptosis is not involved in the mechanism of myocardial dysfunction after resuscitation in a

rat model of cardiac arrest and cardiopulmonary resuscitation. Crit Care Med 2010;38(5):1329-34.

  1. Radhakrishnan J, Ayoub IM, Gazmuri RJ. Activation of caspase-3 may not contribute to postresuscitation myocardial dysfunction. Am J Physiol Heart Circ Physiol 2009;296(4):H1164-74.
  2. Fan J, Cai S, Zhong H, Cao L, Hui K, Xu M, et al. Therapeutic hypothermia attenuates global cerebral reperfusion-induced mitochondrial damage by suppressing dynamin-related protein 1 activation and mitochondria-mediated apoptosis in a cardiac arrest rat model. Neurosci Lett 2017;647:45-52.
  3. Talikowska M, Tohira H, Finn J. Cardiopulmonary Resuscitation quality and patient survival outcome in cardiac arrest: A systematic review and meta- analysis q. Resuscitation 2015;96:66-77.
  4. Cheung PY, Miedzyblocki M, Lee TF. Effects of post-resuscitation administration with sodium hydrosulfide on cardiac recovery in hypoxia- reoxygenated newborn piglets. Eur J Pharmacol 2013;15;718(1-3):74-80.
  5. Riess ML, Matsuura TR, Bartos JA, Bienengraeber M, Aldakkak M, Mcknite SH, et al. Anaesthetic postconditioning at the initiation of CPR improves myocardial and mitochondrial function in a pig model of prolonged untreated ventricular fibrillation. Resuscitation 2014;85(12):1745-51.
  6. Hackenhaar FS, Fumagalli F, Volti GL, Sorrenti V, Russo I, Staszewsky L, et al. Relationship between post-cardiac arrest myocardial oxidative stress and myocardial dysfunction in the rat. J Biomed Sci 2014:21-70.
  7. Anderson TC, Li C-Q, Shao Z-H, Hoang T, Chan KC, Hamann KJ, et al. Transient and partial mitochondrial inhibition for the treatment of postresuscitation injury: getting it just right. Crit Care Med 2006;34. Suppl. (S474-S82).
  8. Sharma AB, Sun J, Howard LL, Williams Jr AG, Mallet RT. Oxidative stress reversibly inactivates myocardial enzymes during cardiac arrest. Am J Physiol Heart Circ Physiol 2007;292(1):H198-206.
  9. Yeh ST, Lee HL, Aune SE, Chen CL, Chen YR, Angelos MG. Preservation of mitochondrial function with cardiopulmonary resuscitation in prolonged cardiac arrest in rats. J Mol Cell Cardiol 2009;47(6):789-97.
  10. Naim MY, Sutton RM, Friess SH, Bratinov G, Bhalala U, Kilbaugh TJ. Blood pressure and coronary perfusion pressure targeted cardiopulmonary resuscitation improves 24-hour survival from ventricular fibrillation cardiac arrest. Crit Care Med 2016;44(11):e1111-7.
  11. Meaney PA, Bobrow BJ, Mancini ME, Christenson J, de Caen AR, Bhanji F, et al. cardiopulmonary resuscitation quality: [corrected] improving cardiac resuscitation outcomes both inside and outside the hospital: a consensus statement from the American Heart Association. Circulation 2013;128 (4):417-35.
  12. Lopaschuk GD, Ussher JR, Folmes CD, Jaswal JS, Stanley WC. Myocardial fatty acid metabolism in health and disease. Physiol Rev 2010;90(1):207-58.
  13. Stanley WC, Recchia FA, Lopaschuk GD. Myocardial substrate metabolism in the normal and failing heart. Physiol Rev 2005;85(3):1093-129.
  14. Katz AM, Reuter H. Cellular calcium and cardiac cell death. Am J Cardiol 1979;44(1):188-90.
  15. Neumar RW, Brown CG, Van LP, Hoekstra J, Altschuld RA, Baker P. Estimation of myocardial Ischemic injury during ventricular fibrillation with total circulatory arrest using high-energy phosphates and lactate as metabolic markers. Ann Emerg Med 1991;20(3):222.
  16. Salcido DD, Menegazzi JJ, Suffoletto BP, Logue ES, Sherman LD. Association of intramyocardial high energy phosphate concentrations with quantitative measures of the ventricular fibrillation electrocardiogram waveform q. Resuscitation 2009;80(8):946-50.
  17. Nakagawa Y, Amino M, Inokuchi S, Hayashi S, Wakabayashi T, Noda T. Novel CPR system that predicts return of spontaneous circulation from amplitude spectral area before Electric shock in ventricular fibrillation. Resuscitation 2017;113:8.
  18. Jaswal JS, Keung W, Wang W, Ussher JR, Lopaschuk GD. Targeting fatty acid and carbohydrate oxidation–a novel therapeutic intervention in the ischemic and failing heart. Biochim Biophys Acta 2011;1813(7):1333-50.

Leave a Reply

Your email address will not be published. Required fields are marked *