Chunhua Ding

Pirfenidone Prevents the Development of a Vulnerable Substrate for Atrial Fibrillation in a Canine Model of Heart Failure

Background— Atrial fibrosis is an important substrate in atrial fibrillation (AF), particularly in the setting of structural heart disease. In a canine model, congestive heart failure (CHF) produces significant atrial fibrosis and the substrate for sustained AF. This atrial remodeling is a potential therapeutic target. The objective of the present study is to evaluate the effects of the antifibrotic drug pirfenidone (PFD) on arrhythmogenic atrial remodeling in a canine CHF model. Methods and Results— We studied 15 canines, divided equally into 3 groups: control, CHF canines not treated with PFD, and CHF canines treated with PFD. CHF was induced by ventricular tachypacing (220 bpm for 3 weeks), and oral PFD was administered for the 3-week pacing period. We performed electrophysiology and AF vulnerability studies, atrial fibrosis measurements, and atrial cytokine expression studies. Only canines in the untreated CHF group developed sustained AF (>30 minutes, 4 of 5 canines; P<0.05). Treatment of CHF canines with PFD resulted in an attenuation of arrhythmogenic left atrial remodeling, with a significant reduction in left atrial conduction heterogeneity index (median [25% to 75% interquartile range] 4.96 [3.53 to 5.64] versus 2.52 [2.11 to 2.82], P<0.01; pacing cycle length 300 ms), left atrial fibrosis (16.0% [13.0% to 17.5%] versus 8.7% [5.7% to 10.6%], P<0.01), and AF duration (1800 [1020 to 1800] seconds versus 6 [5 to 22] seconds, P<0.01). Immunoblotting studies demonstrated the drug’s effects on multiple cytokines, including a reduction in transforming growth factor-&bgr;1 expression. Conclusions— Treatment of CHF canines with PFD results in significantly reduced arrhythmogenic atrial remodeling and AF vulnerability. Pharmacological therapy targeted at the fibrotic substrate itself may play an important role in the management of AF.

Cardiac conduction is required to preserve cardiac chamber morphology

Electrical cardiac forces have been previously hypothesized to play a significant role in cardiac morphogenesis and remodeling. In response to electrical forces, cultured cardiomyocytes rearrange their cytoskeletal structure and modify their gene expression profile. To translate such in vitro data to the intact heart, we used a collection of zebrafish cardiac mutants and transgenics to investigate whether cardiac conduction could influence in vivo cardiac morphogenesis independent of contractile forces. We show that the cardiac mutant dcos226 develops heart failure and interrupted cardiac morphogenesis following uncoordinated ventricular contraction. Using in vivo optical mapping/calcium imaging, we determined that the dco cardiac phenotype was primarily due to aberrant ventricular conduction. Because cardiac contraction and intracardiac hemodynamic forces can also influence cardiac development, we further analyzed the dco phenotype in noncontractile hearts and observed that disorganized ventricular conduction could affect cardiomyocyte morphology and subsequent heart morphogenesis in the absence of contraction or flow. By positional cloning, we found that dco encodes Gja3/Cx46, a gap junction protein not previously implicated in heart formation or function. Detailed analysis of the mouse Cx46 mutant revealed the presence of cardiac conduction defects frequently associated with human heart failure. Overall, these in vivo studies indicate that cardiac electrical forces are required to preserve cardiac chamber morphology and may act as a key epigenetic factor in cardiac remodeling.

Pirfenidone mitigates left ventricular fibrosis and dysfunction after myocardial infarction and reduces arrhythmias

BACKGROUND Post-myocardial infarction (MI) complications include ventricular tachycardia (VT). Excessive non-MI fibrosis, involving the infarct border zone (IBZ) and beyond, is an important substrate for VT vulnerability. OBJECTIVE This study assessed whether the antifibrotic agent pirfenidone can mitigate fibrosis in remodeling and determined its effects on myocardial function and VT susceptibility in a rodent MI model. METHODS We studied 2 groups of rats undergoing MI 1 week prior to treatment: a control group (n = 15) treated with placebo and a pirfenidone group (n = 15). We performed serial echocardiograms, and after 4 weeks of treatment, we conducted electrophysiological and optical mapping studies as well as histology. RESULTS There was less decline in left ventricular (LV) ejection fraction for pirfenidone-treated rats, 8.6% versus 24.3% in controls (P <0.01). Pirfenidone rats also had lower rates of VT inducibility, 28.6% versus 73.3% in control rats (P <0.05). Furthermore, pirfenidone-treated rats had faster conduction velocities in their IBZs compared with controls, at all pacing cycle lengths (P <0.05). Rats treated with pirfenidone also had smaller infarct dense scar (8.9% of LV myocardium vs. 15.7% in controls, P <0.014), less total LV fibrosis (15% vs. 30% in controls, P <0.003), and less nonscar fibrosis (6.6% vs. 12.6% in controls, P <0.006). CONCLUSION Pirfenidone decreased total and nonscar fibrosis in a rat MI model, which correlated with decreased infarct scar, improved LV function, and decreased VT susceptibility. Directly targeting post-MI fibrotic substrates may have a role in limiting infarct-dense scar, improving LV function, and reducing VT vulnerability.

Molecular basis of selective atrial fibrosis due to overexpression of transforming growth factor-β1

AIMS Animal studies show that transforming growth factor-β1 (TGF-β1) is an important mediator of atrial fibrosis and atrial fibrillation (AF). This study investigated the role of TGF-β1 in human AF and the mechanism of atrial-selective fibrosis. METHODS AND RESULTS Atrial specimens from 17 open heart surgery patients and left atrial and ventricular specimens from 17 explanted hearts were collected to assess the relationship between TGF-β1, AF, and differential atrial vs. ventricular TGF-β1 levels. A transgenic mouse model overexpressing active TGF-β1 was used to study the mechanisms underlying the resultant atrial-selective fibrosis. Higher right atrial total TGF-β1 levels (2.58 ± 0.16-fold, P < 0.0001) and active TGF-β1 (3.7 ± 0.7-fold, P = 0.013) were observed in those that developed post-operative AF. Although no ventricular differences were observed, 11 explanted heart failure hearts exhibited higher atrial TGF-β1 levels than 6 non-failing hearts (2.30 ± 0.87 fold higher, P < 0.001). In the transgenic mouse, TGF-β1 receptor-1 kinase blockade resulted in decreased atrial expression of fibrosis-related genes. By RNA microarray analyses in that model, 80 genes in the atria and only 2 genes in the ventricle were differentially expressed. Although these mice atria, but not the ventricles, exhibited increased expression of fibrosis-related genes and phosphorylation of Smad2, there were no differences in TGF-β1 receptor levels or Smads in the atria compared with the ventricles. CONCLUSIONS TGF-β1 mediates selective atrial fibrosis in AF that occurs via TGF-β Receptor 1/2 and the classical Smad pathway. The differential atrial vs. ventricular fibrotic response occurs at the level of TGF-β1 receptor binding or phosphorylation.

High-Resolution Optical Mapping of Ventricular Tachycardia in Rats with Chronic Myocardial Infarction

Background: Ventricular tachycardia (VT) is a common cause of mortality in post‐myocardial infarction (MI) patients, even in the current era of coronary revascularization treatment. We report a reproducible VT model in rats with chronic MI induced by ischemia‐reperfusion and describe its electrophysiological characteristics using high‐resolution optical mapping.

Effects of Tannic Acid, Green Tea and Red Wine on hERG Channels Expressed in HEK293 Cells.

Tannic acid presents in varying concentrations in plant foods, and in relatively high concentrations in green teas and red wines. Human ether-à-go-go-related gene (hERG) channels expressed in multiple tissues (e.g. heart, neurons, smooth muscle and cancer cells), and play important roles in modulating cardiac action potential repolarization and tumor cell biology. The present study investigated the effects of tannic acid, green teas and red wines on hERG currents. The effects of tannic acid, teas and red wines on hERG currents stably transfected in HEK293 cells were studied with a perforated patch clamp technique. In this study, we demonstrated that tannic acid inhibited hERG currents with an IC50 of 3.4 μM and ~100% inhibition at higher concentrations, and significantly shifted the voltage dependent activation to more positive potentials (Δ23.2 mV). Remarkably, a 100-fold dilution of multiple types of tea (green tea, oolong tea and black tea) or red wine inhibited hERG currents by ~90%, and significantly shifted the voltage dependent activation to more positive potentials (Δ30.8 mV and Δ26.0 mV, respectively). Green tea Lung Ching and red wine inhibited hERG currents, with IC50 of 0.04% and 0.19%, respectively. The effects of tannic acid, teas and red wine on hERG currents were irreversible. These results suggest tannic acid is a novel hERG channel blocker and consequently provide a new mechanistic evidence for understanding the effects of tannic acid. They also revealed the potential pharmacological basis of tea- and red wine-induced biology activities.

Immuno‐modification of enhancing stem cells targeting for myocardial repair

Despite the controversy in mechanism, rodent and clinical studies have demonstrated beneficial effects of stem/progenitor cell therapy after myocardial infarction (MI). In a rat ischaemic reperfusion MI model, we investigated the effects of immunomodification of CD 34+ cells on heart function and myocardial conduction. Bispecific antibody (BiAb), consisting of an anti‐myosin light chain antibody and anti‐CD45 antibody, injected intravenously was used to direct human CD34+ cells to injured myocardium. Results were compared to echocardiography guided intramyocardial (IM) injection of CD34+ cells and PBS injected intravenously. Treatment was administered 2 days post MI. Echocardiography was performed at 5 weeks and 3 months which demonstrated LV dilatation prevention and fractional shortening improvement in both the BiAb and IM injection approaches, with BiAb achieving better results. Histological analyses demonstrated a decrease in infarct size and increase in arteriogenesis in both BiAb and IM injection. Electrophysiological properties were studied 5 weeks after treatments by optical mapping. Conduction velocity (CV), action potential duration (APD) and rise time were significantly altered in the MI area. The BiAb treated group demonstrated a more normalized activation pattern of conduction and normalization of CV at shorter pacing cycle lengths. The ventricular tachycardia inducibility was lowest in the BiAb treatment group. Intravenous administration of BiAb offers an effective means of stem cell delivery for myocardial repair post‐acute MI. Such non‐invasive approach was shown to offer a distinct advantage to more invasive direct IM delivery.

Predicting the degree of drug-induced QT prolongation and the risk for torsades de pointes

i o j f a s c QT prolongation can be congenital or acquired. The congenital form is quite rare and is caused by mutations in ion channel subunits or regulatory protein coding genes. Acquired QT prolongation caused by a drug is more prevalent and is often the reason for drug withdrawal from the market or for restriction of use. It is a challenge to predict the risk for individuals to have a QT prolongation or torsades de pointes (Tdp). In this issue of HeartRhythm, Kannankeril et al tried to find the factors affecting the degree of QT prolongation with ibutilide in normal volunteers. To avoid the known factors affecting the QT interval, including hypokalemia, bradycardia, associated heart disease, female gender, dietary salt, and so on, the authors chose healthy volunteers and brought them into salt balance. QTc was measured before and after intravenous administration of ibutilide, a potent blocker of the rapidly activating delayed rectifier potassium current (Ikr). The degree of QTc prolongation ( QTc) by ibutilide as calculated as postibutilide QTc minus baseline QTc. The main finding is that QTc had no correlation to aseline QTc interval. Thus, postdrug QTc was more trongly influenced by QTc than by baseline QTc. Kanankeril et al extrapolated that the risk of TdP is more trongly influenced by QTc than by baseline QTc. Another wo studies were cited to support this. Kaab et al performed acute intravenous sotalol challenge in patients with a history of drug-induced TdP and compared the QT response to control subjects. Those with a history of drug-induced TdP had greater QTc and postdrug QTc, while baseline QTc did not differ. Darbar et al also found that patients with rug-induced long QT syndrome with TdP had higher QTc with similar predrug QTc. But in that study, univarate analysis found QTc was not associated with the deelopment of TdP. So we should be careful not to overemhasize QTc as an index for drug-induced TdP, especially hen the study population is different. Although the QT interval is used as the most practical nd easily measurable parameter of the risk of TdP, it alone

Assessment of cardiac conduction: basic principles of optical mapping.

Extracellular recordings acquired from electrodes placed on the surface of cardiac tissue have traditionally been used to study the electrophysiological properties of the tissue. While this technique has been used in several studies that have increased our understanding of cardiac arrhythmias and action potential propagation, there are several limitations that have prevented us from seeing a bigger picture of arrhythmia mechanisms. These limitations include the limited number of electrodes and unstable recordings. Optical mapping was developed to increase the temporal and spatial resolution over traditional electrode recordings and ultimately the accuracy of the data analysis. This technology involves using a voltage-sensitive dye that binds to the cell membrane. The fluorescence changes of the dye have a linear relationship to the action potential changes of the cell membrane. These fluorescent changes can then be detected by a photodiode array, a CCD camera or a CMOS camera. This will allow the recording of the action potential in hundreds to thousands of different sites simultaneously. Presented in this chapter are the materials and hardware needed along with step-by-step instructions on setup and techniques used in optical mapping for larger tissue preparations.

In vitro Effects of Nerve Growth Factor on Cardiac Fibroblasts Proliferation, Cell Cycle, Migration, and Myofibroblast Transformation

Background: Recent research indicates that nerve growth factor (NGF) promotes cardiac repair following myocardial infarction by promoting angiogenesis and cardiomyocyte survival. The purpose of this study was to investigate the effects of NGF on cardiac fibroblasts (CFs) proliferation, cell cycle, migration, and myofibroblast transformation in vitro. Methods: CFs were obtained from ventricles of neonatal Sprague-Dawley rats and incubated with various concentrations of NGF (0, 0.01, 0.1, 1, 10, and 100 ng/ml; 0 ng/ml was designated as the control group). Cell proliferation and cell cycle of the CFs were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry (FCM), respectively. A cell scratch wound model and transwell were carried out to observe effects of NGF on migration of CFs after 24 h of culture. Real-time polymerase chain reaction (RT-PCR) and Western blotting were used to measure &agr;-smooth muscle actin (&agr;-SMA) at mRNA and protein levels after CFs were incubated with various concentrations of NGF. Results: Expression of &agr;-SMA measured by RT-PCR and Western blotting significantly increased in the 1 and 10 ng/ml NGF groups (P < 0.05). Absorbance values of CFs showed that NGF did not influence the proliferation of CFs (The A490 values were 0.178 ± 0.038, 0.182 ± 0.011, 0.189 ± 0.005, 0.178 ± 0.010, 0.185 ± 0.025, and 0.177 ± 0.033, respectively, in the 0, 0.01, 0.1, 1, 10, and 100 ng/ml NGF groups [P = 0.800, 0.428, 0.981, 0.596, and 0.913, respectively, compared with control group]), and FCM analysis showed that the percentage of CFs in G0/G1, S, and G2/M phases was not changed (P > 0.05). The cell scratch wound model and transwell showed that CFs migration was not significantly different (P > 0.05). Conclusion: NGF induces myofibroblast transformation but does not influence proliferation, cell cycle, or migration of CFs in vitro.