Differential Expression Levels of Integrin α6 Enable the Selective Identification and Isolation of Atrial and Ventricular Cardiomyocytes

Rationale

Central questions such as cardiomyocyte subtype emergence during cardiogenesis or the availability of cardiomyocyte subtypes for cell replacement therapy require selective identification and purification of atrial and ventricular cardiomyocytes. However, current methodologies do not allow for a transgene-free selective isolation of atrial or ventricular cardiomyocytes due to the lack of subtype specific cell surface markers.

Methods and Results

In order to develop cell surface marker-based isolation procedures for cardiomyocyte subtypes, we performed an antibody-based screening on embryonic mouse hearts. Our data indicate that atrial and ventricular cardiomyocytes are characterized by differential expression of integrin α6 (ITGA6) throughout development and in the adult heart. We discovered that the expression level of this surface marker correlates with the intracellular subtype-specific expression of MLC-2a and MLC-2v on the single cell level and thereby enables the discrimination of cardiomyocyte subtypes by flow cytometry. Based on the differential expression of ITGA6 in atria and ventricles during cardiogenesis, we developed purification protocols for atrial and ventricular cardiomyocytes from mouse hearts. Atrial and ventricular identities of sorted cells were confirmed by expression profiling and patch clamp analysis.

Conclusion

Here, we introduce a non-genetic, antibody-based approach to specifically isolate highly pure and viable atrial and ventricular cardiomyocytes from mouse hearts of various developmental stages. This will facilitate in-depth characterization of the individual cellular subsets and support translational research applications.     

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes’ terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.     

Effects of the New Antiarrhythmic Peptide ZP123 on Epicardial Activation and Repolarization Pattern

Antiarrhythmic peptides such as AAP10 (Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH₂) have antiarrhythmic properties related to their stimulatory effect on gap junctional coupling. However, most of these peptides are not stable in enzymatic environment which limits studies with these compounds in vivo. ZP123 is a new antiarrhythmic peptide constructed using a retro-all-D-amino acid design of the AAP10 template (Ac-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH₂). The aim of this study was to compare the effects of AAP10 and ZP123 on epicardial activation and repolarization patterns in isolated perfused rabbit hearts. In addition, we tested the effect of these compounds on PKC activation in cultured HeLa-Cx43 cells. Rabbit hearts were perfused according to the Langendorff technique with Tyrode solution at constant pressure (70 cm H₂O). After 45 min equilibration, either AAP10 (n = 7) or ZP123 (n = 7) was infused intracoronarily in concentrations of 0.1, 1, 10, 100, and 1000 nM (15 min for each concentration) in the presence of 0.05% bovine serum albumine. 256 AgCl electrodes were attached to the hearts surface and connected to the inputs of a 256 channel mapping system in a unipolar circuit (4 kHz/channel, 0.04 mV vertical resolution, 1 mm spatial resolution). For each electrode the activation and repolarization timepoint were determined. We found that both peptides significantly reduced epicardial dispersion by a maximum of about 20% thereby enhancing the homogeneity of epicardial action potential duration, while the action potential duration itself was not affected. The beat-to-beat variability of the epicardial activation pattern was stabilized by both peptides as compared to an untreated time-control series. Other parameters such as LVP, CF, heart rate, or total activation time were not effected by either of the peptides. In a second protocol, rectangular pulses were delivered to the back wall and the propagation velocity was determined longitudinal and transversal to the fiber axis. We found an increase in both longitudinal and transversal conduction velocity. Using a commercial PKC assay on HeLa-Cx43 cells we found that 50 nM AAP10 and 50 nM ZP123 increased activity by 99 ± 6% and 146 ± 54%, respectively. The PKC activation induced by either of these compounds was completely blocked using the selective PKCα inhibitor GCP54345. We conclude that AAP10 and ZP123 have similar effects in vitro, but the superior enzymatic stability of ZP123 makes this compound the preferred substance for in vivostudies of antiarrhythmic peptides.     

Isolation, stages of differentiation, and long term maintenance of adult rat myocytes

Although a variety of techniques have been developed to isolate myocytes from adult hearts, the long term viability of such cells has only recently been investigated. In addition, relatively little is known about the stages of differentiation such cells proceed through following isolation. In the present study myocytes were isolated using two techniques, one involving retrograde perfusion via the aorta, and the other involving mechanical "shearing." In addition, several modifications were made to minimize the trauma normal associated with isolating myocytes from adult hearts. Both techniques yielded a high percentage of rod-shaped, quiescent myocytes, although myocytes isolated using the "shearing" method were less likely to remain viable for more than 24 hours. With both techniques those cells which remained viable for more than 24 hours proceeded through an identical pattern of differentiation leading to stable, attached cells which remained viable for up to four weeks. These results demonstrate that with the appropriate isolation techniques it is possible to maintain adult myocardial cells in culture for lengthy periods of time.     

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (V_m), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both V_m and CaT simultaneously from the same location on the heart^1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping^1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of V_m and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.     

Monoamine oxidase-dependent histamine catabolism accounts for post-ischemic cardiac redox imbalance and injury

Monoamine oxidase (MAO), a mitochondrial enzyme that oxidizes biogenic amines generating hydrogen peroxide, is a major source of oxidative stress in cardiac injury. However, the molecular mechanisms underlying its overactivation in pathological conditions are still poorly characterized.Here, we investigated whether the enhanced MAO-dependent hydrogen peroxide production can be due to increased substrate availability using a metabolomic profiling method. We identified N¹-methylhistamine -the main catabolite of histamine- as an important substrate fueling MAO in Langendorff mouse hearts, directly perfused with a buffer containing hydrogen peroxide or subjected to ischemia/reperfusion protocol. Indeed, when these hearts were pretreated with the MAO inhibitor pargyline we observed N¹-methylhistamine accumulation along with reduced oxidative stress. Next, we showed that synaptic terminals are the major source of N¹-methylhistamine. Indeed, in vivo sympathectomy caused a decrease of N¹-methylhistamine levels, which was associated with a marked protection in post-ischemic reperfused hearts. As far as the mechanism is concerned, we demonstrate that exogenous histamine is transported into isolated cardiomyocytes and triggers a rise in the levels of reactive oxygen species (ROS). Once again, pargyline pretreatment induced intracellular accumulation of N¹-methylhistamine along with decrease in ROS levels. These findings uncover a receptor-independent mechanism for histamine in cardiomyocytes.In summary, our study reveals a novel and important pathophysiological causative link between MAO activation and histamine availability during pathophysiological conditions such as oxidative stress/cardiac injury.     

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.     

Differences in distribution of fibrosis in the ventricles underlie dominant arrhythmia vulnerability of the right ventricle in senescent mice

Mutations that are supposed to affect right (RV) and left ventricular (LV) electrophysiology equally, often reveal dominant conduction slowing and arrhythmia vulnerability in RV. In this study we investigated the mechanism of dominant arrhythmia vulnerability of RV in senescent mice. We performed epicardial ventricular activation mapping on adult and senescent Langendorff perfused hearts. Longitudinal and transversal conduction velocity, as well as arrhythmia inducibility were determined. Subsequently, hearts were processed for immunohisto-chemistry and Picro Sirius Red staining. Senescent mice revealed decreased conduction velocity, increased aniso-tropic ratio and reduced excitation wavelength in RV, but not in LV. Arrhythmias were mainly induced in RV of senescent hearts. No arrhythmias were induced in adult hearts. Immunohistochemistry revealed that the amount of Connexin 43 and cardiac sodium channel Nav1 .5 were equally decreased, and that collagen content was equally increased in senescent RV and LV. However, patches of replacement fibrosis were found throughout the RV wall, but only in the sub-endocardium and mid-myocardium of LV. The study shows that the dominant arrhythmia vulnerability in RV of senescent mice is caused by the distribution of replacement fibrosis which involves the entire RV but only part of the LV. (Neth Heart J 2008; 16:356-8.)     

Effects of the new antiarrhythmic peptide ZP123 on epicardial activation and repolarization pattern

Antiarrhythmic peptides such as AAP10 (Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH(2)) have antiarrhythmic properties related to their stimulatory effect on gap junctional coupling. However, most of these peptides are not stable in enzymatic environment which limits studies with these compounds in vivo. ZP123 is a new antiarrhythmic peptide constructed using a retro-all-D-amino acid design of the AAP10 template (Ac-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH(2)). The aim of this study was to compare the effects of AAP10 and ZP123 on epicardial activation and repolarization patterns in isolated perfused rabbit hearts. In addition, we tested the effect of these compounds on PKC activation in cultured HeLa-Cx43 cells. Rabbit hearts were perfused according to the Langendorff technique with Tyrode solution at constant pressure (70 cm H(2)O). After 45 min equilibration, either AAP10 (n = 7) or ZP123 (n = 7) was infused intracoronarily in concentrations of 0.1, 1, 10, 100, and 1000 nM (15 min for each concentration) in the presence of 0.05% bovine serum albumine. 256 AgCl electrodes were attached to the hearts surface and connected to the inputs of a 256 channel mapping system in a unipolar circuit (4 kHz/channel, 0.04 mV vertical resolution, 1 mm spatial resolution). For each electrode the activation and repolarization timepoint were determined. We found that both peptides significantly reduced epicardial dispersion by a maximum of about 20% thereby enhancing the homogeneity of epicardial action potential duration, while the action potential duration itself was not affected. The beat-to-beat variability of the epicardial activation pattern was stabilized by both peptides as compared to an untreated time-control series. Other parameters such as LVP, CF, heart rate, or total activation time were not effected by either of the peptides. In a second protocol, rectangular pulses were delivered to the back wall and the propagation velocity was determined longitudinal and transversal to the fiber axis. We found an increase in both longitudinal and transversal conduction velocity. Using a commercial PKC assay on HeLa-Cx43 cells we found that 50 nM AAP10 and 50 nM ZP123 increased activity by 99 +/- 6% and 146 +/- 54%, respectively. The PKC activation induced by either of these compounds was completely blocked using the selective PKCalpha inhibitor GCP54345. We conclude that AAP10 and ZP123 have similar effects in vitro, but the superior enzymatic stability of ZP123 makes this compound the preferred substance for in vivo studies of antiarrhythmic peptides.     

Use of endogenous NADH fluorescence for real-time in situ visualization of epicardial radiofrequency ablation lesions and gaps

Radiofrequency ablation (RFA) aims to produce lesions that interrupt reentrant circuits or block the spread of electrical activation from sites of abnormal activity. Today, there are limited means for real-time visualization of cardiac muscle tissue injury during RFA procedures. We hypothesized that the fluorescence of endogenous NADH could be used as a marker of cardiac muscle injury during epicardial RFA procedures. Studies were conducted in blood-free and blood-perfused hearts from healthy adult Sprague-Dawley rats and New Zealand rabbits. Radiofrequency was applied to the epicardial surface of the heart using a 4-mm standard blazer ablation catheter. A dual camera optical mapping system was used to monitor NADH fluorescence upon ultraviolet illumination of the epicardial surface and to record optical action potentials using the voltage-sensitive probe RH237. Epicardial lesions were seen as areas of low NADH fluorescence. The lesions appeared immediately after ablation and remained stable for several hours. Real-time monitoring of NADH fluorescence allowed visualization of viable tissue between the RFA lesions. Dual recordings of NADH and epicardial electrical activity linked the gaps between lesions to postablation reentries. We found that the fluorescence of endogenous NADH aids the visualization of injured epicardial tissue caused by RFA. This was true for both blood-free and blood-perfused preparations. Gaps between NADH-negative regions revealed unablated tissue, which may promote postablation reentry or provide pathways for the conduction of abnormal electrical activity.     

成年大鼠心肌细胞分离培养及兴奋-收缩耦联表征

目的：比较两种细胞分离液分离成年大鼠心肌细胞,进一步表征成年大鼠心室肌细胞兴奋-收缩耦联。方法 Langendorff装置进行主动脉逆流灌流,分别用两种细胞分离液分离成年大鼠心肌细胞,无血清培养并进行腺病毒感染。显微镜下观察单个心肌细胞的形态学特点,荧光显微镜下检测病毒感染。采用IonOptix仪器检测心肌细胞肌节收缩-舒张指标以及心肌细胞钙离子摄入-排出指标。结果两种分离液均可获得70％横纹清晰的长杆状心肌细胞,培养可存活7 d以上。腺病毒感染48 h,绿色荧光蛋白持续表达7 d以上。分离液一获得的心肌细胞不能很好地随电场刺激产生收缩,分离液二获得的细胞可用于检测兴奋-收缩耦联特性,心肌细胞肌节缩短分数为11.61％±2.15％,舒张时间为（0.177±0.031） s,钙瞬变幅度为30.79％±9.74％,钙瞬变衰减时间为（0.300±0.074） s。结论两种分离液均可用于分离和培养成年大鼠心肌细胞,并用于腺病毒转染等长时程研究。分离液二更适用于检测成年大鼠心肌细胞的兴奋-收缩耦联特性。     

成年小鼠心肌细胞分离技术

为进行成年小鼠心肌细胞培养与收缩功能研究, 首先必须获得高产量与高质量的心肌细胞。本实验采用Langendorff装置行恒流灌流心脏, 同时监测灌流压力的变化。根据小鼠鼠龄微调灌流流速, 使初始灌流压力保持在40 mmHg。用0.05 % 单一粗制胶原酶在37 ℃条件下消化心脏, 当灌流压力下降至28 mmHg 时, 即刻终止消化。轻轻吹散心肌细胞后, 用含1 % 牛血清白蛋白的 Joklik’s MEM 培养液保存,逐步法恢复细胞外钙离子浓度。获得的心肌细胞存活率大于 70 %,复钙后耐钙心肌细胞静置 4 h,心肌细胞存活率仍能保持在(40~50) %。其中,90 %以上存活的长杆状心肌细胞无明显搏动,细胞膜表面光滑,横纹清晰,两端边缘锐利, 折光性较强, 复钙后保存4 h 或 5.0 Hz 刺激5 min 后,仍能保持正常形态。在1.0 Hz刺激条件下, 心肌细胞缩短幅度为(9.72 ±0.43) %; 2.0 Hz 刺激下为(11.28 ±0.43) %; 在5.0 Hz 刺激下, 达到(11.40 ±0.45)%。这些结果表明, 采用本方法可获得高产量与高质量的成年小鼠心肌细胞, 且易于操作, 重复性较好。     

ATPase inhibitory factor 1 protects the heart from acute myocardial ischemia/reperfusion injury through activating AMPK signaling pathway

Rationale: Myocardial ischemia/reperfusion (I/R) injury is a common clinic scenario that occurs in the context of reperfusion therapy for acute myocardial infarction (AMI). The mitochondrial F1Fo-ATPase inhibitory factor 1 (IF1) blocks the reversal of the F1Fo-ATP synthase to prevent detrimental consumption of cellular ATP and associated demise. In the present study, we study the role and mechanism of IF1 in myocardial I/R injury.Methods: Mice were ligated the left anterior descending coronary artery to build the I/R model in vivo. Rat hearts were isolated and perfused with constant pressure according to Langendorff. Also, neonatal cardiomyocytes hypoxia-reoxygenation (H/R) model was also used. Myocardial infarction area, cardiac function, cellular function, and cell viability was conducted and compared.Results: Our data revealed that IF1 is upregulated in hearts after I/R and cardiomyocytes with hypoxia/re-oxygenation (H/R). IF1 delivered with adenovirus and adeno-associated virus serotype 9 (AAV9) ameliorated cardiac dysfunction and pathological development induced by I/R ex vivo and in vivo. Mechanistically, IF1 stimulates glucose uptake and glycolysis activity and stimulates AMPK activation during in vivo basal and I/R and in vitro OGD/R conditions, and activation of AMPK by IF1 is responsible for its cardioprotective effects against H/R-induced injury.Conclusions: These results suggest that increased IF1 in the I/R heart confer cardioprotective effects via activating AMPK signaling. Therefore, IF1 can be used as a potential therapeutic target for the treatment of pathological ischemic injury and heart failure.     

Repression of Cyclin D1 Expression Is Necessary for the Maintenance of Cell Cycle Exit in Adult Mammalian Cardiomyocytes

The hearts of neonatal mice and adult zebrafish can regenerate after injury through proliferation of preexisting cardiomyocytes. However, adult mammals are not capable of cardiac regeneration because almost all cardiomyocytes exit their cell cycle. Exactly how the cell cycle exit is maintained and how many adult cardiomyocytes have the potential to reenter the cell cycle are unknown. The expression and activation levels of main cyclin-cyclin-dependent kinase (CDK) complexes are extremely low or undetectable at adult stages. The nuclear DNA content of almost all cardiomyocytes is 2C, indicating the cell cycle exit from G₁-phase. Here, we induced expression of cyclin D1, which regulates the progression of G1-phase, only in differentiated cardiomyocytes of adult mice. In these cardiomyocytes, S-phase marker-positive cardiomyocytes and the expression of main cyclins and CDKs increased remarkably, although cyclin B1-CDK1 activation was inhibited in an ATM/ATR-independent manner. The phosphorylation pattern of CDK1 and expression pattern of Cdc25 subtypes suggested that a deficiency in the increase in Cdc25 (a and -b), which is required for M-phase entry, inhibited the cyclin B1-CDK1 activation. Finally, analysis of cell cycle distribution patterns showed that >40% of adult mouse cardiomyocytes reentered the cell cycle by the induction of cyclin D1. The cell cycle of these binucleated cardiomyocytes was arrested before M-phase, and many mononucleated cardiomyocytes entered endoreplication. These data indicate that silencing the cyclin D1 expression is necessary for the maintenance of the cell cycle exit and suggest a mechanism that involves inhibition of M-phase entry.     

Cardiac Regeneration from Activated Epicardium

In contrast to lower vertebrates, the mammalian heart has a very limited regenerative capacity. Cardiomyocytes, lost after ischemia, are replaced by fibroblasts. Although the human heart is able to form new cardiomyocytes throughout its lifespan, the efficiency of this phenomenon is not enough to substitute sufficient myocardial mass after an infarction. In contrast, zebrafish hearts regenerate through epicardial activation and initiation of myocardial proliferation. With this study we obtain insights into the activation and cellular contribution of the mammalian epicardium in response to ischemia. In a mouse myocardial infarction model we analyzed the spatio-temporal changes in expression of embryonic epicardial, EMT, and stem cell markers and the contribution of cells of the Wt1-lineage to the infarcted area. Though the integrity of the epicardial layer overlaying the infarct is lost immediately after the induction of the ischemia, it was found to be regenerated at three days post infarction. In this regenerated epicardium, the embryonic gene program is transiently re-expressed as well as proliferation. Concomitant with this activation, Wt1-lineage positive subepicardial mesenchyme is formed until two weeks post-infarction. These mesenchymal cells replace the cardiomyocytes lost due to the ischemia and contribute to the fibroblast population, myofibroblasts and coronary endothelium in the infarct, and later also to the cardiomyocyte population. We show that in mice, as in lower vertebrates, an endogenous, epicardium-dependent regenerative response to injury is induced. Although this regenerative response leads to the formation of new cardiomyocytes, their number is insufficient in mice but sufficient in lower vertebrates to replace lost cardiomyocytes. These molecular and cellular analyses provide basic knowledge essential for investigations on the regeneration of the mammalian heart aiming at epicardium-derived cells.     

Effects of heart isolation,voltage-sensitive dye,and electromechanical uncoupling agents on ventricular fibrillation

We tested whether the interventions typically required for optical mapping affect activation patterns during ventricular fibrillation (VF). A 21 x 24 unipolar electrode array (1.5 mm spacing) was sutured to the left ventricular epicardium of 16 anesthetized pigs, and four episodes of electrically induced VF (30-s duration) were recorded. The hearts were then rapidly excised and connected to a Langendorff perfusion apparatus. Four of the hearts were controls, in which 24 additional VF episodes were then mapped. In the remaining 12 hearts, four VF episodes were mapped after isolation, four more episodes were mapped after exposure to the voltage-sensitive dye di-4-ANEPPS, and six more episodes were mapped after exposure to the electromechanical uncoupling agents diacetyl monoxime (DAM; 20 mmol/l, n = 6) or cytochalasin D (CytoD; 10 micromol/l, n = 6). VF episodes were separated by 4 min. VF activation patterns were quantified using custom pattern analysis algorithms. From comparisons with time-corrected control data, all interventions significantly changed VF patterns. Most changes were broadly consistent with slowing and regularization due to loss of excitability. Heart isolation had the largest effect on VF patterns, followed by CytoD, DAM, and dye.     

Culture and adenoviral infection of adult mouse cardiac myocytes: methods for cellular genetic physiology

Rapid development of transgenic and gene-targeted mice and acute genetic manipulation via gene transfer vector systems have provided powerful tools for cardiovascular research. To facilitate the phenotyping of genetically engineered murine models at the cellular and subcellular levels and to implement acute gene transfer techniques in single mouse cardiomyocytes, we have modified and improved current enzymatic methods to isolate a high yield of high-quality adult mouse myocytes (5.3 +/- 0.5 x 10(5) cells/left ventricle, 83.8 +/- 2.5% rod shaped). We have also developed a technique to culture these isolated myocytes while maintaining their morphological integrity for 2-3 days. The high percentage of viable myocytes after 1 day in culture (72.5 +/- 2.3%) permitted both physiological and biochemical characterization. The major functional aspects of these cells, including excitation-contraction coupling and receptor-mediated signaling, remained intact, but the contraction kinetics were significantly slowed. Furthermore, gene delivery via recombinant adenoviral infection was highly efficient and reproducible. In adult beta(1)/beta(2)-adrenergic receptor (AR) double-knockout mouse myocytes, adenovirus-directed expression of either beta(1)- or beta(2)-AR, which occurred in 100% of cells, rescued the functional response to beta-AR agonist stimulation. These techniques will permit novel experimental settings for cellular genetic physiology.     

Optical mapping of cryoinjured rat myocardium grafted with mesenchymal stem cells

Mesenchymal stem cells (MSCs) have been shown to improve cardiac electrophysiology when administered in the setting of acute myocardial infarction. However, the electrophysiological phenotype of MSCs in situ is not clear. We hypothesize that MSCs delivered intramyocardially to cryoinjured myocardium can engraft, but will not actively generate, action potentials. Cryoinjury-induced scar was created on the left ventricular epicardial surface of adult rat hearts. Within 30 min, hearts were injected with saline (sham, n = 11) or bone marrow-derived MSCs (2 × 10(6)) labeled with 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanine percholate (DiI; n = 16). At 3 wk, optical mapping and cell isolation were used to measure optical action potentials and calcium transients, respectively. Histological analysis confirmed subepicardial scar thickness and the presence of DiI-positive cells that express connexin-43. Optical action potential amplitude within the scar at MSC-positive sites (53.8 ± 14.3%) was larger compared with sites devoid of MSCs (35.3 ± 14.2%, P < 0.05) and sites within the scar of shams (33.5 ± 6.9%, P < 0.05). Evidence of simultaneous action potential upstroke, the loss of action potential activity following ablation of adjacent viable myocardium, and no rapid calcium transient response in isolated DiI+ cells suggest that the electrophysiological influence of engrafted MSCs is electrotonic. MSCs can engraft when directly injected into a cryoinjury and are associated with evidence of action potential activity. However, our results suggest that this activity is not due to generation of action potentials, but rather passive influence coupled from neighboring viable myocardium.     

Mapping of reentrant spontaneous polymorphic ventricular tachycardia in a Scn5a+/- mouse model

Two major mechanisms have been postulated for the arrhythmogenic tendency observed in Brugada Syndrome (BrS): delays in conduction or increased heterogeneities in repolarization. We use a contact mapping system to directly investigate the interacting roles of these two mechanisms in arrhythmogenesis using a genetic murine model for BrS for the first time. Electrograms were obtained from a multielectrode recording array placed against the left ventricle and right ventricle (RV) of spontaneously beating Langendorff-perfused wild type (WT) and Scn5a+/- mouse hearts. Scn5a+/- hearts showed activation waves arriving at the epicardial surface consistent with slowed conduction, which was exacerbated in the presence of flecainide. Lines of conduction block across the RV resulting from premature ventricular beats led to the formation of reentrant circuits and polymorphic ventricular tachycardia. WT hearts showed an inverse relationship between activation times and activation recovery intervals measured at the epicardial surface, which resulted in synchronicity of repolarization times. In contrast, Scn5a+/- hearts, despite having smaller mean activation recovery intervals, demonstrated a greater heterogeneity compared with WT. Isochronal maps showed that their normal activation recovery interval gradients at the epicardial surface were disrupted, leading to heterogeneity in repolarization times. We thus directly demonstrate the initiation of arrhythmia in the RV of Scn5a+/- hearts. This occurs as a result of the combination of repolarization heterogeneities leading to lines of conduction block and unidirectional conduction, with conduction slowing allowing the formation of reentrant circuits. The repolarization heterogeneities may also be responsible for the changing pattern of block, leading to the polymorphic character of the resulting ventricular tachycardia.