Dyslipidemia and Cardiac Ventricular Arrhytmias

Background
Hypercholesterolemia (HC) is a major threat to public health in modern society and has been considered as a primary risk factor for cardiovascular diseases. Lipid-lowering interventions have been shown to reduce coronary events and all-cause mortality. It is possible that some of the beneficial effects of lipid-lowering therapy can be attributed to the reduction of ventricular tachycardia/fibrillation (VT/VF) and sudden death. Mitchell et al sought to evaluate the anti-arrhythmic effects of lipid-lowering therapy in patients enrolled in Antiarrhythmics Versus Implantable Defibrillators (AVID) trial with larger patient population. In patients with atherosclerotic heart disease (ASHD) and implantable cardiovertor defibrillator (ICD), the use of lipid-lowering drug therapy was associated with a 40% reduction in the relative hazard for VT/VF recurrence. However, it is still unclear why these lipid-lowering agents should act on the arrhythmic substrate.
Tissue heterogeneity, exacerbated by electrical and structural remodeling from cardiac disease, has traditionally been considered the major factor promoting wavebreak and its degeneration to fibrillation. Recently, however, dynamic factors have also been recognized to play a key role. Dynamic factors refer to cellular properties of the cardiac action potential and calcium cycling, which dynamically generate wave instability and wavebreak. Electrical dynamic factors include restitution of action potential duration (APD) and conduction velocity (CV), short-term cardiac memory, and electrotonic currents. Calcium dynamic factors are related to dynamic calcium cycling properties. They act synergistically, as well as with tissue heterogeneity, to promote wavebreak and fibrillation. In addition to electrical remodeling, Chen’s group performed several studies to determine the relationship between nerve sprouting and cardiac arrhythmia. It is clear that, in addition to the electrical, structural, and mechanical remodeling documented in numerous models of
atrial and ventricular arrhythmias, one has to consider neural remodeling. The neurogenic factors of the arrhythmia seemed to play as important a role as
myogenic factors.
The majority of sudden deaths are attributed to VF, often triggered by acute ischemic events. Primary VF in acute myocardial infarction predicted higher short-term mortality. Successful resuscitation in this situation is extremely difficult, resulting in a dismal survival rate. The mechanisms by which the usual cardiopulmonary resuscitation efforts fail in patients with acute regional ischemia are not well understood. Clarifying the mechanisms by which VF occurs during acute ischemia should be an important step towards more efficient prevention and/or treatment of sudden cardiac death. It is well known that myocardial ischemia alters the regional electrophysiological properties by depolarizing the cellular membrane, reducing membrane excitability, shortening APD, slowing CV, and prolonging the refractoriness beyond repolarization. Both APD restitution and excitability are important in the maintenance of VF. Prior study has classified VF into two types. The type 1 VF is associated with a steep APDR, normal excitability and multiple wavelets. The type 2 VF is associated with a flat APD restitution, low excitability, broad CV restitution, and spatiotemporal periodicity with intermittent wavebreaks. During acute regional ischemia, the ischemic region produced by acute occlusion of coronary artery is characterized by both flattening of APDR and reduced excitability, while the non-ischemic region retains normal excitability and steep APDR. Thus, both types of VF may co-exist during early phase of regional ischemia, resulting in increased difficulties in electrical defibrillation and resuscitation. Regional ischemic model can demonstrate the alterations in the ischemic and adjacent non-ischemic zone simultaneously and can help us to understand the interaction between the ischemic and non-ischemic cardiac tissue, which should be important for clarifying the mechanism of sudden cardiac death.
The broad and long-term objective of this research project is to test the hypothesis that dyslipidemia increases the susceptibility of life-threatening arrhythmia underlies the mechanisms of cardiac arrhythmogenesis and sudden cardiac death in HC patients. In the traditional view, HC could result in atherosclerosis, endothelial dysfunction and plaque instability in coronary arteries, predisposing myocardial ischemia and/or infarction. It provides both triggers and substrate (tissue heterogeneity) for cardiac arrhythmia. The importance of interaction between ischemic and non-ischemic regions in initiation and maintenance of VF should be further explored. We also think that hypercholesterolemia could have direct effects on the myocardium. We further hypothesized that the interaction between neural remodeling (nerve sprouting) and electrical remodeling of myocardium induced by hypercholesterolemia underlies the mechanisms of arrhythmogenesis. In addition to coronary arteries, myocardium also severed as a target organ of serum cholesterol. Myocardial ischemia, neural remodeling and electrophysiological remodeling all contribute to the pro-arrhythmic effects of hypercholesterolemia. Meanwhile, the clinical significance of dyslipidemia on ventricular arrhythmia will also be evaluated in the clinical setting of acute coronary syndrome. The Specific Aims of this research project are as follows:
Specific Aim 1 is to test the hypothesis that dyslipidemia increased the risk of developing ventricular tachyarrhythmia in the clinical setting of acute myocardial infarction.
Specific Aim 2 is to test the hypothesis that HC can induce proarrhythmic neural and electrophysiological remodeling in the heart, increasing the susceptibility of VF.
Specific Aim 3 is to test the hypothesis that regional ischemic tissue could result in co-existing 2 type of VF in a heart, increasing vulnerability and complexity of VF during acute coronary occlusion.

Materials and methods
In Specific Aim 1, a total of 58 patients experiencing VT/VF within 24 hours after the onset of MI were matched to 58 patients with MI but without VT/VF. Sex (104 males), age (58 ± 10 years), and the use of thrombolytic therapy (n=82) were matched in both groups initially. The lipid profiles including total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglyceride were measured during the first week and at the 3rd month after the index MI. Other coronary risk factors, clinical, hemodynamic, and angiographic characteristics were also included in our assessment.
In Specific Aim 2, We fed rabbits with either high cholesterol (HC, N=10, 6, 15, 25) or standard (S, N=10, 6, 15, 20) chows for 12 weeks (Protocol 1,4-6), and with HC (N=12) or S (N=10) chows for 8 weeks (Protocol 2). In Protocol 3, 10 rabbits were fed with various protocols to observe the effects of different serum cholesterol levels. Twelve-lead electrocardiograms were recorded for QTc and QTc dispersion analysis very 2 weeks during the feeding period in Protocol 1. Electrophysiological study and optical mapping of Langendorff- perfusion hearts were performed at the end of feeding period in Protocol 1 and 2. The alterations in APD, APD dispersion, CV and vulnerability of VF were studied. We used immunocytochemical stain of GAP-43 (a maker for nerve sprouting) and TH (a maker of sympathetic nerve) to evaluated sympathetic activity “in vitro” in Protocol 1 and 3; and used myocardial 123I-meta- iodobenzylguanidine (123I-MIBG) imaging to evaluated sympathetic activity “in vivo” in Protocol 4. Whole-cell clamp recordings of calcium inward currents and protein expression of Na+/Ca2+ exchanger (NCX) L-type Ca channel dihydropyridine receptor (DHPR), and sarcoplasmic reticulum Ca2+-ATPase (SERCA) were studied to explore alterations in calcium handling in HC rabbit hearts in Protocol 5. Immunoblotting and immuofluorescence microscopic study of connexin 43 (Cx43) were also done to see the remodeling of gap junction in HC rabbits hearts in Protocol 6.
In Specific Aim 3, optical mapping studies were performed with di-4ANEPPS in 15 Langendorff-perfused rabbit hearts. Branches of coronary artery were ligated to create regional ischemia in 10 hearts. APD measured to 50% repolarization (APD50) during ischemia showed an area with uniformly shortened APD50 (Zone 1), normal or lengthened APD50 (Zone 3) and an area in between with an APD50 gradient (Zone 2). APD restitution, CV and VF dynamics were analyzed in different areas (Zone 1, 2, and 3) of regional ischemic hearts. We further determined whether Type I and Type II VF could be demonstrated in heart with regional ischemia and the importance of interaction between 2 types of VF on VF complexity.

Results:
In Specific Aim 1, patients with VT/VF had a higher level of LDL-C and a lower blood pressure on the initial arrival during the acute myocardial infaction. At the 3-month follow up, those patients with VT/VF had a higher level of TC, LDL-C and triglyceride. In multivariate analysis, the LDL-C (p< 0.001) at the 3-month follow up, the mean blood pressure on arrival (p< 0.01), and the difference in the triglyceride levels between the first week and the third month (p< 0.05) were all the independent predictors for the occurrence of VT/VF in the acute stage of MI. The relative risk of occurrence of VT/VF was 3.89 for each increasing in 50 mg/dl of LDL cholesterol (95% CI: 1.74-8.69), 1.54 for each increasing in 50 mg/dl of the difference between 1-week and 3-month triglyceride (95% CI: 1.01-2.34), and 0.44 for each increasing in 20 mmHg of mean blood pressure (95% CI: 0.26-0.75). The 3-month serum LDL cholesterol was the most significant independent predictor of the occurrence of VT/VF during the first day of acute MI.
In Specific Aim 2, the serum cholesterol levels were 2097 ± 288 mg/dl in HC group and 59 ± 9 mg/dl in S group for Protocol 1 and were 1889 ± 577 mg/dl in HC group and 50 ± 21 mg/dl in S group for Protocol 2. In Protocol 4 and 5, the serum cholesterol (3022 ±158 mg/dl vs. 42 ±4 mg/dl; p<0.001) levels were also significantly higher in HC group by the end of 3-month period. Density of GAP43- and TH-positive nerves in the heart were significantly higher in HC (5587 ± 3747 and 2608 ± 2592 μm2/mm2) than S (2165 ± 1443 and 462 ± 687μm2/mm2, respectively, p<0.001) in Protocol 1. Protocol 3 showed a strong correlation between serum cholesterol level and nerve density for GAP43 (R2= 0.94; p<0.001) and TH (R2= 0.91; p<0.001). The cardiac MIBG uptake of HC group, which was evaluated with heart/ mediastinum (H/M) ratio, was higher (3.39 ± 0.11 vs. 2.90 ± 0.10; p<0.05) than that of the age-matched control group. Compared with S, HC rabbits had longer QTc intervals (311 ±10 ms vs. 287 ± 9 ms, p=0.001), more QTc dispersion (63.5 ± 13 ms vs. 18.5 ± 4 ms, p<0.001), longer APD (At pacing cycle length, PCL=400 ms;APD80: 193 ± 21 ms vs. 174 ± 17 ms; p<0.05) and increased heterogeneity of repolarization (At PCL=400 ms; SD: 8.7 ± 2.9 ms vs. 5.5 ± 2.0 ms, p<0.01; difference: 44.7 ± 16.9 ms vs. 25.1 ± 7.3 ms, p<0.01) in Protocol 1 and 2. The measured CVs for HC rabbits were also significantly less than that of the control (At PCL=300 ms; 52.88 ± 1.45 cm/s versus 59.11±1.64 cm/s, P<0.001). Ventricular fibrillation was either induced or occurred spontaneously in 9/12 of hearts of HC group and 2/10 of hearts in S group in Protocol 2. The peak L-type calcium inward current density was significantly higher in HC myocytes (14.0 ± 3.1 pA/pF vs. 9.1 ± 3.4 pA/pF, p<0.01). Western blot experiments revealed that protein expression of SERCA in the HC strips decreased (p<0.05), but that of the NCX increased (p<0.05) in Protocol 5. The protein expression of the DHPR was similar between these two groups. Western blotting demonstrated a 21% reduction of connexin43 (Cx43) protein expression within three weeks. Along the feeding protocol for 12 weeks, expression of cardiomyocyte Cx43 protein progressively decreased (60% reduction at 12 weeks, P<0.001) in Protocol 6. Such a reduction was also demonstrated by immunoconfocal microscopy with decreases in the proportion of total tissue area occupied by Cx43 signals and the size/density of Cx43 gap-junctional plaques. Withdrawing a cholesterol- enriched diet for 18 weeks restored the Cx43 protein expression.
In Specific Aim 3, ischemia flattened APD restitution slope (lowest max. slope; baseline: 1.01 ± 0.52, 30 min of ischemia: 0.56 ± 0.32, 60 min of ischemia: 0.55±0.30; p<0.01) and reduced conduction velocity in ischemic zone (Zone 1), creating a condition for type 2 VF. APD restitution steepened (highest max. slope; baseline: 1.65 ± 0.70, 30 min of ischemia: 2.35 ± 0.71, 60 min of ischemia: 2.59 ± 1.04; p<0.01) and the conduction velocity changed little in the non-ischmic zone (Zone 3), creating a condition for type 1 VF. During induced VF, the dominant frequency in Zones 2 and 3 progressively increased after the onset of ischemia. The dominant frequency in Zone 1 (ischemic zone) first decreased and then slightly increased, but typically remained less than the dominant frequency in the Zone 3. We also analyzed the spread of activation into and out of the ischemic region by mapping activation sequences in those VF episodes at 30 min of ischemia. We found about 22% of wavefronts propagating from ischemic zone to non-ischemic zone, 41% of wavefronts propagating from non-ischemic zone to ischemic zone, and 37% of waves collided near the ischemic border zone. The number of wavebreaks increased with time in all 3 zones (baseline, 4.3 ± 1.5; 30 min 11.7 ± 5.6; 60 min 15.6 ± 11/frame; p<0.01).

Conclusion:
In the clinical setting of acute myocardial infarction, our study suggests that dyslipidemia imposes a higher risk of developing tachyarrhythmia in the acute phase of myocardial infarction. It is possible that the baseline serum lipid and the triglyceride levels during acute phase of myocardial infarction played a significant role in the arrhythmogenesis. In the rabbit model of HC, HC induced significant nerve sprouting and sympathetic hyperinnervation in both immunocytochemical studies “in vitro” and 123I -MIBG imaging “in vivo”. Abnormal calcium handling (increased ICa, decreased SERCA protein expression, and increased NCX protein level) and down-regulation of connexin 43 were also demonstrated in heart with HC. The neural and electrophysiological remodeling was associated with prolonged APD, longer QTc intervals, increased repolarization dispersion, decreased CV and increased ventricular vulnerability to fibrillation. In regional ischemic rabbit heart model, two types of VF can co-exist during acute regional ischemia. Both ischemic (decreased excitability) and non-ischemic regions (steepen APD restitution) developed proarrhythmic changes during regional ischemia, therefore, contributing to the increased ventricular vulnerability to VF and sudden death during acute coronary occlusion. The findings in both HC rabbit model and regional ischemic model provided mechanistic insight into the dyslipidemia predisposing to the occurrence of VT/VF during acute stage of myocardial infarction. Meanwhile, these results may also partially explain the beneficial effects of lipid-lowering drugs in reducing the risk of sudden cardiac death in large-scale clinical trials.
In spite of recent advances in cardiovascular medicine, sudden cardiac death remains a major public health problem. In addition to anatomical and electrical remodeling, neural remodeling also plays a role in the generation of sudden cardiac death. The mechanism by which β-blocker reduces the risk of arrhythmia has been unclear. Likewise, the dramatic antiarrhythmic effect of statin remains an outstanding puzzle. In contrast to the poor outcome of drugs targeting ion channel function, it appears that autonomic control may become a new and effective target for the management of cardiac rhythm. Such a view is supported by a large body of recent evidence showing that neural remodeling as manifested by sympathetic hyperinnervation and nerve sprouting exists in diseased hearts. Due to the significant health implications of HC in humans, it is necessary to further study the effects of HC induced neural and electrical remodeling on potential risk of cardiac arrhythmias. We will conduct future studies to study the interaction between electrical remodeling and neural remodeling using different animal models. The additional knowledge obtained from future project will further extend the understanding of the mechanisms of sudden cardiac death, and potentially lead to new therapeutic approaches for the management of heart rhythm.