Molecular Genetics of Lidocaine-containing Cardioplegia in the Human Heart during Cardiac Surgery

Abstract

Background:

During cardiac surgery with cardiopulmonary bypass, delivery of cardioplegia solution to achieve electromechanical cardiac quiescence is obligatory. The addition of lidocaine to cardioplegia has advantages, however, its consequences at a molecular level remain unclear. We performed whole-genome RNA sequencing of the human left ventricular (LV) myocardium to elucidate the differences between whole blood (WB) cardioplegia with and without addition of lidocaine (LC) on gene expression.

Methods:

We prospectively enrolled 130 patients undergoing aortic valve replacement surgery. Patients received high-potassium blood cardioplegia either with (n=37) or without (n=93) lidocaine. The LV apex was biopsied at baseline, and after an average of 74 minutes of cold cardioplegic arrest. We performed differential gene expression analysis for 18,258 genes between these two groups. Clinical and demographic variables were adjusted in the model. Gene ontology (GO) and network enrichment analysis of the retained genes were performed using g:Profiler and Cytoscape.

Results:

1,298 genes were differentially expressed between cardioplegic treatments. Compared to the WB group, genes upregulated in the LC group were identified by network enrichment to play a protective role in ischemic injury by inhibiting apoptosis, increasing transferrin endocytosis, and increasing cell viability. Down-regulated genes in the LC group were identified to play a role in inflammatory diseases, oxygen transport, and neutrophil aggregation.

Conclusions:

The addition of lidocaine to cardioplegia had significant effects on a molecular level with genes responsible for decreased inflammation, reduced intracellular calcium binding, enhanced anti-apoptotic protection, augmented oxygen accessibility through transferrins, and increased cell viability showing measurable differences.

Intra-coronary cardioplegia solution is used during cardiac surgery with cardiopulmonary bypass (CPB) to cause cessation of cardiac activity to facilitate a relaxed, bloodless field for surgical lesion repair. This lowers the metabolic rate of the heart muscle to prevent cell death during aortic cross clamping with cardioplegic arrest. Multiple cardioplegia delivery methods and solutions have been used, with the current technique at many institutions being a cold hyperkalemic whole-blood solution that rapidly depolarizes the myocardium and readily wears off.

Cardioplegic solutions prevent the generation and propagation of action potentials and subsequent contractions by providing a high concentration of potassium ions into the extracellular space. Hyperpolarizing cells during ischemia with potassium also limits intracellular Ca²+ accumulation which has been shown to increase myocardial injury during reperfusion.(1) However, not all cardioplegic solutions are equally efficacious at controlling intracellular Ca²+ in the myocardium. One technique to control Ca²+ accumulation is the addition of lidocaine to the cardioplegia solution. Lidocaine, a class Ib Na+ channel blocker, prevents circulatory Na+ influx into cells, thereby indirectly limiting Ca²+ accumulation during ischemia.(2) In pediatric cardiac surgery, studies have shown decreased myocardial injury with the use of lidocaine-containing cardioplegia.(3) Due to its favorable outcomes in pediatric cardiac surgery, it has also been studied in the aging myocardium, with resultant decreased troponin release, superior myocardial functional recovery, lower insulin requirements and time and cost savings.(4–7) In our own center, we demonstrated that the use of a whole-blood cardioplegia containing lidocaine (LC) compared to propensity-matched controls receiving whole-blood cardioplegia was safe with similar postoperative outcomes (WB).(8) However, despite good clinical outcomes, the effect of LC cardioplegia on the aging human myocardium at a molecular level using gene expression information has not been studied.

Therefore, in this prospective, single-center, single-surgeon study, we examined whole-genome differential gene expression signatures at baseline and after a prolonged period of cardioplegic arrest in patients with a traditional WB cardioplegia solution compared to LC cardioplegia. We hypothesized that alterations in gene expression could explain discrepancies in outcome studies, and guide us in future cardioplegia use.

Material and Methods

Study Population

We prospectively enrolled 130 patients undergoing elective aortic valve replacement surgery with cardiopulmonary bypass (CPB) at a single institution from 2009–2015 (https://clinicaltrials.gov/ct2/show/NCT00985049). 50% of the patients also had concomitant coronary artery bypass grafting surgery (CABG). Patients received our standard WB cardioplegia solution (N=94), detailed below, up until May 30^th, 2013, at which time we switched to LC cardioplegia solution (n=37). We made the switch to add lidocaine to our regular cardioplegia solution after learning of the many favorable reported advantages, which include reduced cross-clamp time, decreased transfusion rates, decreasing the risk of reperfusion injury, and limiting the amount of intracellular calcium accumulation during cardioplegic arrest.(4–7, 9)

Cardioplegia techniques

Patients were systemically cooled to mild hypothermia using a bladder temperature of 34 to 35 degrees Celsius. A left ventricular decompression vent (Medtronic 10f pediatric vent) was placed in the left ventricular apex. Cold antegrade cardioplegia with and without lidocaine was delivered at 8 to 12 degrees Celsius after aortic cross clamp placement via the aortic root. Delivered volume was indexed at 10 ml/kg body weight at a rate of approximately 200 ml/min. Antegrade cardioplegia was delivered until a constant asystolic arrest was achieved. At the discretion of the surgeon, if a retrograde cardioplegia catheter (Medtronic DLP 15f auto inflate) was placed in the coronary sinus, the delivery route was changed to retrograde until the indexed dose was completed. The surgeon was notified after 40 minutes had elapsed after the initial delivery or at any time electrical activity appeared on the ECG. At the surgeon’s discretion, additional dose(s) of our institution’s standard low potassium cardioplegia may have been delivered retrograde through the coronary sinus or antegrade directly through the coronary ostia.

Cardioplegia Solutions

Our institution’s standard cardioplegia formula consists of 8 parts blood and 1 part crystalloid/electrolyte admixture (Table 1). The initial arresting dose admixture consists of 70mEq potassium chloride and 18.68 mEq magnesium in 250 ml normal saline (Central Admixture Pharmacy Services, Inc., Lehigh Valley, PA), giving a delivered concentration of approximately 30mEq potassium and 8.3 mEq magnesium per liter. For additional cardioplegia doses, our maintenance cardioplegia formula is identical to our standard formula except it has a reduced delivered potassium concentration of approximately 10 mEq per liter and is delivered on average every 20 minutes. For the LC cardioplegia, 225 mg of 10% lidocaine was added to our arresting dose admixture allowing for a delivered concentration 100 mg lidocaine per liter. Any additional doses were given with our standard maintenance cardioplegia without the addition of lidocaine to avoid a prolonged period of electrical arrest after aortic cross clamp removal.

Table 1.

Standard Cardioplegia and Lidocaine-containing cardioplegia

Delivered Dose Per Liter
Cardioplegia	Lidocaine in admixture	No Lidocaine in admixture	Del Nido	St. Thomas II (Plegisol)
Carrier	0.9% NaCl	0.9% NaCl	Plasmalyte	0.9% NaCl
Blood to crystalloid ratio	8:1	8:1	1:4	0:1
Mannitol 20% (grams)	-	-	1.6	-
Sodium bicarbonate (mEq)	-	-	10.4	-
Sodium Chloride (mEq)	148	148	-	120
Magnesium Sulfate (mEq)	8	8	12.8	32
Potassium Chloride (mEq)	30	30	16.5	16
Lidocaine (mg)	100	-	104	-
Calcium (mEq)	-	-	-	2.4

mEq = milliequivalent

Sample collection:

Procedures were performed in accordance with the ethical standards of The Partners Healthcare Institutional Review Board, which approved this study, and written informed consent was obtained from each patient for the tissue biopsy. Punch biopsies were obtained from the site of the routinely placed left ventricular decompression vent in the anterolateral apical wall of the left ventricle (LV) before the onset of ischemia (baseline sample) and again shortly before removal of the aortic cross-clamp (post-ischemia) after approximately 80 minutes of cardioplegic arrest. A total of ~3–5 μg total RNA content from the ventricular myocardium was collected. The transition from WB to LC cardioplegia was a change in practice pattern and therefore did not require an additional consent. Collected tissue samples were placed in RNAlater® (Ambion, ThermoFisher Scientific, Waltham, MA) within 1–2 minutes, and were kept at +4°C for 48 hours and subsequently stored at −80°C until RNA extracti on.

RNA Sequencing and Computational Analyses.:

RNA sequencing and the computational analysis for gene expression analysis and gene enrichment analysis has been previously described and a detailed description can be found in Supplement 1.(10–12) Briefly, differential expression analysis between the two groups was performed, adjusted for age, gender, cross-clamp-time (CCT), and batch effects, and p-values were corrected for multiple comparisons.

Subsequently, we performed gene ontology (GO) and network enrichment analysis of the genes whose expression at post-ischemia differed between cardioplegic treatments.

Results

Patients receiving LC cardioplegia (N=37) were slightly younger, had a higher ejection fraction and were less likely to have hypercholesterolemia. As intended, they also received a smaller volume of total cardioplegia, had less re-doses, and a longer time until the first re-dose. No difference existed between groups regarding sex, BMI, diabetes, aortic cross-clamp-time, or mode of cardioplegia delivery for any doses (antegrade versus retrograde versus both). Demographic and clinical variables of the study are presented in Table 2.

Table 2.

Demographic and clinical variables

Patient demographics	Whole Blood Cardioplegia (N=93)	Lidocaine containing Cardioplegia (N=37)	P- value
Female Gender - N (%)	37 (40%)	11 (29%)	0.13
Age (years)	72 ± 11	66 ± 13	0.02
Body mass index (kg/m2)	30 ± 7	31 ± 7	0.46
Diabetes - N (%)	36 (39%)	11 (29%)	0.40
Left Ventricle Ejection Fraction (%)	56 ± 12	60 ± 7	0.04
Hypercholesterolemia- N (%)	70 (75%)	22 (59%)	0.04
Clinical Variables
Total Volume of Cardioplegia (ml)	1678 ± 533	1290 ± 438	<0.001
Aortic Cross-Clamp-time (min)	84 ± 29	77 ± 28	0.21
More than 1 re-dose of cardioplegia -N (%)	33 (36%)	2 (6%)	<0.001
Average time to re-dose (min)	25 ± 6	36 ± 10	<0.001
Antegrade cardioplegia	6 (7%)	2 (6%)	0.87
Retrograde cardioplegia	8 (9%)	2 (6%)
Antegrade & retrograde cardioplegia	76 (84%)	29 (88%)

Expressed as mean ± standard deviation.

Differential gene expression

At the post-ischemia time point, 1,298 genes were significantly differentially expressed between LC and WB cardioplegia. Of those, 48 genes were found to be significantly differentially expressed between post-ischemia and baseline (Figure 1). These 48 genes (12 up-regulated and 36 down-regulated) were therefore identified as genes whose expression differed between type of cardioplegia as a result of ischemia. Up-regulated genes of interest (Table 3, Figure 2a) are involved in the determination of cell fate [SRY-related HMG-box) 18 (SOX18)], protein binding and dimerization [Kelch Like Family Member 38 (KLHL38)], adenosine regulation [5’ Nucleotidaze Cytosolic 1A (NT5C1A)], and connecting transmembrane proteins to the actin cytoskeleton and G-protein-coupled signaling pathways [SH3 And Multiple Ankyrin Repeat Domains 3 (SHANK3)]. Down-regulated genes (Table 3, Figure 2b) play a role in the regulation of the inflammatory process [S100 Calcium Binding Protein A9 and A9 (S100A8–9)], and have been associated with atherosclerosis, vasculitis, and other inflammatory disorders. In addition, several genes appear to attenuate oxidative stress resulting from ischemic injury. All 36 down-regulated genes are presented in Supplement 2.

Figure 1.

Volcano plot of top up & down differentially expressed genes for whole-blood cardioplegia (WB) versus lidocaine cardioplegia (LC); each dot represents a gene; Red-dots represent genes with an adjusted p-value < 0.05; Orange-dots represent genes with log₂ fold change > 0.5; Green-dots represent genes for both an adjusted p-value < 0.05 and a log₂ fold change > 0.5.

Table 3.

Top 20 up- and down regulated genes comparing whole-blood (WB) with lidocaine (LC) cardioplegia.

Gene symbol	Gene name	Locus	Log2 fold change	P-value	Adjusted P-value	Mean expression (WB)	Mean expression (LC)
SOX18	SOX (SRY- related HMG- box) 18	20q13.33	0.91	1.0E-03	1.1E-02	5.81	6.93
KLHL38	Kelch Like Family Member 38	8q24.13	0.88	1.0E-03	8.0E-03	4.68	5.68
NT5C1A	5’ Nucleotidaze Cytosolic 1A	1p34.2	0.82	3.5E-04	4.3E-03	2.68	3.27
CCDC85B	Coiled-Coil Domain Containing 85B	11q13.1	0.81	5.9E-06	3.0E-04	6.46	7.34
SHANK3	SH3 And Multiple Ankyrin Repeat Domains 3	22q13.33	0.80	7.2E-05	1.0E-03	4.97	6.01
HES4	Hes Family BHLH Transcription Factor 4	1p36.33	0.79	2.0E-03	1.4E-02	4.62	5.69
ODZ2	Odd Oz/Ten-M Homolog 2	5q34	0.74	1.0E-03	8.7E-03	4.89	5.30
METRN	Meteorin, Glial Cell Differentiation Regulator	16p13.33	0.66	3.0E-04	3.0E-03	5.07	5.90
XAF1	XIAP Associated Factor	17p13.2	0.63	9.9E-06	5.0E-04	4.40	4.98
KANK3	KN Motif And Ankyrin Repeat Domains 3	19p13.2	0.63	5.0E-03	2.9E-02	4.63	5.69
S100A9	S100 Calcium Binding Protein A9	1q21.3	−1.59	3.6E-04	4.3E-03	5.42	3.76
S100A8	S100 Calcium Binding Protein A8	1q21.3	−1.58	6.7E-04	6.5E-03	4.63	2.82
HBB	Hemoglobin Subunit Beta Chain	11p15.4	−1.32	7.2E-03	3.4E-02	9.65	8.10
S100A12	S100 Calcium Binding Protein A12	1q21.3	−1.13	2.0E-03	1.4E-02	2.53	1.22
CCL21	C-C Motif Chemokine Ligand 21	9p13.3	−1.12	9.1E-03	4.1E-02	3.26	2.25
IFI27	Interferon Alpha Inducible Protein 27	14q32.12	−1.07	6.9E-06	4.2E-04	7.18	6.41
COX4I2	Cytochrome C Oxidase Subunit IV Isoform 2	20q11.21	−1.04	1.3E-05	6.1E-04	4.19	3.35
RBP7	Retinol Binding Protein 7	1p36.22	−0.97	5.3E-04	5.5E-03	5.23	4.52
CLEC3B	C-Type Lectin Domain Family 3 Member B	3p21.31	−0.95	2.8E-04	3.7E-03	7.13	6.22
FPR1	Formyl Peptide Receptor 1	19q13.41	−0.93	4.3E-04	4.8E-03	2.16	1.39

Gene expression values are shown as mean (log2) based on FPKM (fragments per kilobase of transcript per million mapped reads). Adjusted P-value = adjusted for multiple comparisons using the Benjamini-Hochberg algorithm; P-value= probability significant difference between gene expression of two groups for the specific gene.

Figure 2.

(a) Comparison of mean gene-expression between whole-blood cardioplegia (WB) versus whole-blood with lidocaine cardioplegia (LC) for top ten up-regulated genes. Genes expression are expressed as means, with 25^th and 75^th percentiles (shown by vertical dimension of the box), and the 10^th and 90^th percentiles indicated by the whiskers. (b) Comparison of mean gene-expression between whole-blood cardioplegia (WB) versus whole-blood with lidocaine cardioplegia (LC) for top ten down-regulated genes. Genes expression are expressed as means, with 25^th and 75^th percentiles (shown by vertical dimension of the box), and the 10^th and 90^th percentiles indicated by the whiskers.

Functional classification

To understand the biological function of significant genes, we examined the 48 differentially expressed genes for enrichment of GO categories in g:Profiler, and subsequently investigated network enrichment in CytoScape. We identified 12 significant signaling pathways using gene set enrichment analysis (GSEA). To explore whether these 12 sets reflect a common biological function, we examined the leading-edge subset for each gene set. The leading-edge subset consist of 2 genes (S100A8, S100A9), encoding products involved into the “neutrophil aggregation” signaling pathway. This shared subset in the GSEA signal of the 12 gene sets points to down-regulation of this component of the neutrophil aggregation pathway as a key distinction between the LC and WB cardioplegia. Genes with significant enrichment in a specific GO category are presented in Table 4 and visualized in Figure 3.

Table 4.

Pathway analysis using gene set enrichment of significant differential genes when comparing whole blood cardioplegia with and without the addition of lidocaine.

Gene ontology #	Gene-set	Gene-set Description	Gene number in set	P-value
GO:0050786	S100B,S100A12,S100A4,FPR1,HMGB1,S100A9,S100A13,S10 0A8,S100P, HMGB2, S100A7	RAGE receptor binding	11	2.11E-08
GO:0070488	S100A9,S100A8	neutrophil aggregation	2	1.72E-05
GO:0035662	TIRAP,S100A9,DAB2IP,S100A8	Toll-like receptor 4 binding	4	1.02E-04
GO:0005504	PPARG,PTGDS,FABP5,ID3,S100A9,NDUFAB1,UGT1A8,SNC A,ALOX5AP,ARHGDIA,STX3,APOC1,ALB,PMP2,S100A8,HNF 4A,FABP4,RIDA,ADH5,OXER1,SCP2,FABP1,PPARD,UCP1,F ABP3,NME2,FABP2,SH3GLB1,FFAR4	fatty acid binding	29	1.2E-04
GO:0050544	PPARG,ALOX5AP,STX3, S100A9,S100A8	arachidonic acid binding	5	1.7E-04
GO:0050832	ANG,TGFB1,HAMP,CHGA,S100A9,DEFA6,IL36RN,COTL1,HT N3,NLRP10,CLEC6A,S100A8,ADM,IL17A,LTF,IL17RA,DEFA1 B,S100A12IL17RC,JAGN1,DEFA1,DEFA4,GNLY,ELANE,MPO, HTN1,HRG,C10ORF99,SPON2,DCD,CTSG,DEFA5,DEFA3	defense response to fungus	33	1.8E-04
GO:1901567	PPARG,ALOX5AP,STX3,S100A9,S100A8,OXER1	fatty acid derivative binding	6	2.2E-04
GO:0050542	PPARG,ALOX5AP,STX3,S100A9,S100A8,OXER1	icosanoid binding	6	2.5E-04
GO:0050543	PPARG,ALOX5AP,STX3,FABP3,S100A9,S100A8	icosatetraenoic acid binding	6	2.5E-04
GO:0009620	ANG,TGFB1,SYK,HAMP,CLEC7A,BAK1,CHGA,DEFA6,S100A 9,COTL1,IL36RN,HTN3,NLRP10,MYD88,CLEC6A,ADM,IL17A, S100A8,LTF,IL17RA,DEFA1B,S100A12,IL17RC,DEFA1,JAGN1 ,DEFA4,GNLY,CD86,MALT1,PTX3,ELANE,MPO,IL6,HTN1,IL2 5,HRG,BTK,C10ORF99,CHIA,CTSG,CARD9,DEFA5,DCD,SPO N2,BCL 10,TLR4,DEFA3	response to fungus	47	5.1E-04
GO:0035325	TIRAP,TLR6,SYK,TOLLIP,TLR1,TLR2,S100A9,UNC93B1,DAB 2IP,S100A8	Toll-like receptor binding	10	7.5E-04
GO:0036041	PPARG,RIDA,ALOX5AP,PPARD,UCP1,FABP3,STX3,S100A9, S100A8,SCP2,OXER1	long-chain fatty acid binding	11	9.2E-04

Gene-set= group of genes that make a pathway for a biological function

Figure 3.

Gene-set enrichment analysis. This figure shows enrichment of specific functions (GO terms) by interactions between functions. Nodes represent gene-sets and edges represent mutual overlap. Each circle (red) represents a biological function of a specific gene-set containing at least one gene. Each node (green-line) represents connectivity between these gene-sets. The yellow circle (RAGE) represents the highest significant gene-set enrichment (P=2.11E-08).

Comment

The effect of different types of cardioplegia in humans on a molecular level has not been examined. In this study, we show that LC cardioplegia has the potential to favorably change the expression of genes involved in myocardial protection for patients undergoing cardiac surgery. When using LC cardioplegia for CPB, we identified differentially expressed genes and GO pathways that attenuate ischemic injury to the myocardium. Among functions, these genes are associated with improved intracellular calcium control, anti-apoptosis of myocardium cells, and reduction in oxidative stress.

Cardioplegia solutions containing lidocaine were originally developed for pediatric cardiac surgery by Dr. del Nido over twenty years ago.(13) The clinical benefits of lidocaine-containing cardioplegia solutions are that a single-dose strategy avoids the interruption of the flow of surgery and thereby significantly reduces cross-clamp time,(14) while lowering of peak glucose values and insulin requirements,(5) decreased transfusion rates,(15) and a decreased rate of postoperative atrial fibrillation(8) have also been reported. Lidocaine polarizes myocardial cell membranes to prevent sodium and, indirectly, calcium accumulation within the cells, thereby decreasing the risk of reperfusion injury. Lidocaine inhibits the fast voltage gated sodium (Na) channels on myocyte membranes, decreasing the Na current window during cardioplegic arrest. With a reduction in sodium influx into the cell, L-type Ca channels are less activated and reverse mode Na+/Ca²+ exchange is decreased. Thus, lidocaine limits the amount of intracellular calcium accumulation that ultimately contributes to ischemia-reperfusion injury during cardioplegic arrest.(4)

Down-regulated genes

The two most significant down-regulated genes (S100A9, S100A8) form the Calprotectin protein complex, which are part of the Damage-Associated Molecular Pattern molecules (DAMPs) released during tissue damage.(16) These genes encode calcium and zinc binding proteins, which play a prominent role in the regulation of the inflammatory process. This complex can induce cell death via autophagy and apoptosis.(17) Furthermore, Calprotectin is a well-known marker of inflammatory conditions and its elevation has been associated with atherosclerosis, vasculitis, and other inflammatory disorders.(18). The down-regulation of this complex suggests that the myocardium protected by LC may experience a reduction in the inflammatory response as well as cell death.

Another highly down-regulated gene in the LC group was hemoglobin subunit beta (HBB). HBB is a protein-coding gene involved in oxygen transport and can also be expressed in non-erythroid cell types.(19) A rapid increase of HBB expression has been observed in the right ventricular myocardium in infants and children with congenital heart disease in response to ischemia, and in isolated cardiomyocytes during simulated ischemia reperfusion injury.(20) Furthermore, HBB expression has been shown to increase due to oxidative stress.(19) Therefore, a decrease of HBB expression as seen in the LC group, may signal less oxidative stress from ischemic injury.

COX4I2, which encodes the terminal enzyme (Cytochrome C oxidative, CcOx) of the mitochondrial respiratory chain, plays a pivotal role in cytochrome C regulation and its related pathways, which include cardiac muscle contraction and respiratory electron transport.(21) The lower expression of this gene after LC cardioplegia and ischemia suggests a reduced rate of cellular respiration, leading to an overall reduction in metabolic activity of the myocardium.(22) In hypoxic conditions, CcOx activity is increased and thereby plays an important role in the generation of reactive oxygen species resulting in ischemic injury.(23) Similar to HBB, a reduced expression of COX4I2 might be a sign of less oxidative stress from ischemic injury.

Up-regulated genes

The most up-regulated gene in the LC group compared to the WB cardioplegia group was SOX18, which is a transcription factor involved in the determination of cell fate. It also plays a role in blood vessel and lymphatic vessel development,(24) and has been associated with atherosclerosis.(25) Given the short period of cardioplegic arrest, it is unlikely that SOX18 upregulation in the LC group represents angiogenesis, but rather is a sign of early cellular differentiation and growth. Garcia-Ramirez et al has shown that inhibition of SOX18 expression decreases both DNA synthesis and vascular cell growth,(25) and thus warrants further investigation as a protective agent.

5’ Nucleotidaze Cytosolic 1A (NT5C1A), also upregulated in the LC group, regulates adenosine levels in the heart during ischemia and hypoxia.(26) The nucleoside adenosine is an important regulator within the myocardium as these G-protein coupled receptors modulate coronary flow, heart rate and contraction, and have an effect on cardiac protection, inflammatory regulation, and control of cell growth and tissue remodeling.(27) With its protective potential the adenosine receptor system is an intriguing therapeutic target as it limits cardiac injury, suppresses inflammation, and restores the balance between myocardial energy utilization and supply. The increased expression of this gene after lidocaine containing cardioplegia give insight into a possible protective mechanism.

Gene ontology (GO) and biological function

Eleven downregulated genes formed a significant network and biological process related to the receptor for advanced glycation end products (RAGE) inhibitor. The RAGE on the cell membrane is hypothesized to have a causative effect in a range of inflammatory diseases and has been linked to several chronic diseases resulting from vascular damage, such as myocardial infarction and heart failure.(16) In cardiovascular disease, advanced glycation end products can induce crosslinking of collagen, which can cause vascular stiffening and entrapment of low-density lipoprotein particles (LDL) in the arterial wall.(28).

Neutrophil aggregation has been directly implicated in pathophysiologic setting such as those found during cardiovascular ischemic episodes. The 2 genes in this pathway (S100A8, S100A9) may limit the potential harmful effects of aggregated neutrophils on vascular endothelium, which have been directly linked to ischemic complications in cardiovascular settings.

Another significant enrichment of genes, all of which were down-regulated in the LC group, was related to Toll-like receptor 4 (TLR4) in functional analysis. TLR4 is a transmembrane protein, and its activation leads to an intracellular signaling pathway, NF-kB, and inflammatory cytokine production, which is responsible for activating the innate immune system.(29) During ischemia-reperfusion injury, TLR4 and IL-1 receptor-associated kinase (IRAK)-1 activation is suppressed.(30) IL-10 has been suggested to help maintain heart function during stress via myeloid differentiation gene 88/IRAK-4/IRAK-1-dependent TLR4 signaling.(30)

Limitations

Our study has several strengths, including having been carried out with human heart tissue rather than simulated sequence data or an animal model. It is also, to the best of our knowledge, the first evaluation of cardioplegic solutions during heart surgery at the molecular level using RNA-seq data in a human model. However, this is a retrospective, single-institution, single surgeon study and is subject to all limitations inherent to such a design. The addition of lidocaine was a change in practice and reflected clinical care, which could have varied from patient to patient, though we adjusted for these variances with statistical modeling. We are also limited in our observations to a single type of valve surgery, though half of the patients did also have CABG surgery.

Conclusions

In summary, we showed that the addition of lidocaine to cardioplegia significantly changes gene expression in the human LV. While we cannot definitely state that these changes are protective, genes responsible for decreased inflammation, reduced intracellular calcium binding, enhanced anti-apoptotic protection, augmented oxygen accessibility through transferrins, and increased cell viability show measurable differences. However, further investigation of the biological significance is prudent.