Mitochondrial Networks in Cardiac Myocytes Reveal Dynamic Coupling Behavior

Oscillatory behavior of mitochondrial inner membrane potential (ΔΨ_m) is commonly observed in cells subjected to oxidative or metabolic stress. In cardiac myocytes, the activation of inner membrane pores by reactive oxygen species (ROS) is a major factor mediating intermitochondrial coupling, and ROS-induced ROS release has been shown to underlie propagated waves of ΔΨ_m depolarization as well as synchronized limit cycle oscillations of ΔΨ_m in the network. The functional impact of ΔΨ_m instability on cardiac electrophysiology, Ca²⁺ handling, and even cell survival, is strongly affected by the extent of such intermitochondrial coupling. Here, we employ a recently developed wavelet-based analytical approach to examine how different substrates affect mitochondrial coupling in cardiac cells, and we also determine the oscillatory coupling properties of mitochondria in ventricular cells in intact perfused hearts. The results show that the frequency of ΔΨ_m oscillations varies inversely with the size of the oscillating mitochondrial cluster, and depends on the strength of local intermitochondrial coupling. Time-varying coupling constants could be quantitatively determined by applying a stochastic phase model based on extension of the well-known Kuramoto model for networks of coupled oscillators. Cluster size-frequency relationships varied with different substrates, as did mitochondrial coupling constants, which were significantly larger for glucose (7.78 × 10⁻² ± 0.98 × 10⁻² s⁻¹) and pyruvate (7.49 × 10⁻² ± 1.65 × 10⁻² s⁻¹) than lactate (4.83 × 10⁻² ± 1.25 × 10⁻² s⁻¹) or β-hydroxybutyrate (4.11 × 10⁻² ± 0.62 × 10⁻² s⁻¹). The findings indicate that mitochondrial spatiotemporal coupling and oscillatory behavior is influenced by substrate selection, perhaps through differing effects on ROS/redox balance. In particular, glucose-perfusion generates strong intermitochondrial coupling and temporal oscillatory stability. Pathological changes in specific catabolic pathways, which are known to occur during the progression of cardiovascular disease, could therefore contribute to altered sensitivity of the mitochondrial network to oxidative stress and emergent ΔΨ_m instability, ultimately scaling to produce organ level dysfunction.     

低氧与心肌细胞凋亡

细胞凋亡是心肌细胞低氧损伤的主要死亡形式之一。低氧引起心肌细胞凋亡可以通过外部的死亡受体通路以及内部的线粒体通路,两条通路之间又存在复杂的交互作用,其中,线粒体通路在低氧诱导的心肌细胞凋亡中起重要作用。另外,心肌细胞本身也具有多种内源性的凋亡抑制因子。因此,低氧时心肌细胞凋亡的产生是多种因素综合作用的结果,Bcl-2家族蛋白、线粒体通透性改变、细胞色素c的释放以及caspases的活化等参与了低氧引起的心肌细胞凋亡的调控。对低氧时心肌细胞凋亡的认识和深入研究,为人类在缺血性心脏病的防治中提供了一个新的治疗措施。     

Arrhythmogenic Transient Dynamics in Cardiac Myocytes

Cardiac action potential alternans and early afterdepolarizations (EADs) are linked to cardiac arrhythmias. Periodic action potentials (period 1) in healthy conditions bifurcate to other states such as period 2 or chaos when alternans or EADs occur in pathological conditions. The mechanisms of alternans and EADs have been extensively studied under steady-state conditions, but lethal arrhythmias often occur during the transition between steady states. Why arrhythmias tend to develop during the transition is unclear. We used low-dimensional mathematical models to analyze dynamical mechanisms of transient alternans and EADs. We show that depending on the route from one state to another, action potential alternans and EADs may occur during the transition between two periodic steady states. The route taken depends on the time course of external perturbations or intrinsic signaling, such as β-adrenergic stimulation, which regulate cardiac calcium and potassium currents with differential kinetics.     

Mechanotransduction in Stretch-Induced Hypertrophy of Cardiac Myocytes

Abstract

Mechanical loading of cardiac muscles causes rapid activation of a number of immediate-early (IE) genes and hypertrophy. However, little is known as to how muscle cells sense mechanical load and regulate gene expression. We examined roles of several putative mechanotransducers in stretch-induced hypertrophy of cardiac myocytes grown on a deformable silicone sheet. Using the patch-clamp technique, we found a single class of stretch-activated cation channels which was completely and reversibly blocked by gadolinium. The inhibition of this channel by gadolinium did not affect either stretch-induced expression of the IE genes or hypertrophy. Neither disruption of microtubules with colchicine nor that of actin microfilaments by cytochalasin D prevented the stretch-induced IE gene expression. Arresting contractile activity by tetrodotoxin did not affect the stretch-induced IE gene expression or hypertrophy. These results suggest that stretch-activated cation channels, microtubules, microfilaments, and contractile activity are not the mechanotransducers. Preliminary results suggest that cell stretch may cause a release of a growth factor(s), which in turn initiates a cascade of hypertrophic response of cardiac myocytes.     

Phosphoregulation of the Titin-cap Protein Telethonin in Cardiac Myocytes

Telethonin (also known as titin-cap or t-cap) is a muscle-specific protein whose mutation is associated with cardiac and skeletal myopathies through unknown mechanisms. Our previous work identified cardiac telethonin as an interaction partner for the protein kinase D catalytic domain. In this study, kinase assays used in conjunction with MS and site-directed mutagenesis confirmed telethonin as a substrate for protein kinase D and Ca²⁺/calmodulin-dependent kinase II in vitro and identified Ser-157 and Ser-161 as the phosphorylation sites. Phosphate affinity electrophoresis and MS revealed endogenous telethonin to exist in a constitutively bis-phosphorylated form in isolated adult rat ventricular myocytes and in mouse and rat ventricular myocardium. Following heterologous expression in myocytes by adenoviral gene transfer, wild-type telethonin became bis-phosphorylated, whereas S157A/S161A telethonin remained non-phosphorylated. Nevertheless, both proteins localized predominantly to the sarcomeric Z-disc, where they partially replaced endogenous telethonin. Such partial replacement with S157A/S161A telethonin disrupted transverse tubule organization and prolonged the time to peak of the intracellular Ca²⁺ transient and increased its variance. These data reveal, for the first time, that cardiac telethonin is constitutively bis-phosphorylated and suggest that such phosphorylation is critical for normal telethonin function, which may include maintenance of transverse tubule organization and intracellular Ca²⁺ transients.     

Pharmacological Inhibition of AMP-Deaminase in Rat Cardiac Myocytes

Because mutation of AMP deaminase 1 gene leading to reduced AMP deaminase activity may result in protection of cardiac function in patients with heart disease, inhibitors of AMP deaminase (AMPD) may have therapeutic applications. This study evaluated the effect of a specific inhibitor of AMP deaminase 3-[2-(3-carboxy-4-bromo-5,6,7,8-tetrahydronaphthyl)ethyl]-3,6,7,8-tetrahydroimidazo [4,5-d][1,3]diazepin-8-ol (AMPDI) on the isolated human enzyme and on nucleotide catabolism in rat cardiomyocytes. AMPDI effectively inhibited isolated human AMPD with an IC ₅₀ = 0.5 μ M. AMPDI was much less effective with isolated cardiomyocytes (IC ₅₀ = 0.5 mM). AMPDI is a very effective inhibitor of AMPD that despite lower efficiency in the cell system examined could be useful for in vivo studies.     

Circadian Rhythms of Early Afterdepolarizations and Ventricular Arrhythmias in a Cardiomyocyte Model

Sudden cardiac arrest is a malfunction of the heart’s electrical system, typically caused by ventricular arrhythmias, that can lead to sudden cardiac death (SCD) within minutes. Epidemiological studies have shown that SCD and ventricular arrhythmias are more likely to occur in the morning than in the evening, and laboratory studies indicate that these daily rhythms in adverse cardiovascular events are at least partially under the control of the endogenous circadian timekeeping system. However, the biophysical mechanisms linking molecular circadian clocks to cardiac arrhythmogenesis are not fully understood. Recent experiments have shown that L-type calcium channels exhibit circadian rhythms in both expression and function in guinea pig ventricular cardiomyocytes. We developed an electrophysiological model of these cells to simulate the effect of circadian variation in L-type calcium conductance. In our simulations, we found that there is a circadian pattern in the occurrence of early afterdepolarizations (EADs), which are abnormal depolarizations during the repolarization phase of a cardiac action potential that can trigger fatal ventricular arrhythmias. Specifically, the model produces EADs in the morning, but not at other times of day. We show that the model exhibits a codimension-2 Takens-Bogdanov bifurcation that serves as an organizing center for different types of EAD dynamics. We also simulated a two-dimensional spatial version of this model across a circadian cycle. We found that there is a circadian pattern in the breakup of spiral waves, which represents ventricular fibrillation in cardiac tissue. Specifically, the model produces spiral wave breakup in the morning, but not in the evening. Our computational study is the first, to our knowledge, to propose a link between circadian rhythms and EAD formation and suggests that the efficacy of drugs targeting EAD-mediated arrhythmias may depend on the time of day that they are administered.     

The Rate- and Species-Dependence of Short-Term Memory in Cardiac Myocytes

Short-term memory is an intrinsic property of paced cardiac myocytes that reflects the influence of pacing history, and not just the immediately preceding diastolic interval (DI), on the action potential duration (APD). Although it is recognized that short-term memory affects the dynamics of cardiac myocytes in general, and the onset of irregular cardiac rhythm in particular, its has never been adequately quantified or measured directly in experiments or numerical simulations, mainly due to the absence of appropriate techniques. As a result, very little is known about the rate- and species dependent behavior of short-term memory. In this study, we introduce a new approach that allows one to estimate how much short-term memory, M _S, is present in the cardiac myocyte at different pacing rates. The new quantification is based on the fact that pacing history affects not only the APD, but the entire dynamics of paced cardiac myocytes, in particular the restitution curve. Using the patch clamp technique and numerical simulations, we measured short-term memory restitution—the dependence of M _S on the cycle length—in isolated rabbit and guinea pig ventricular myocytes. In both species, M _S is rate- and species-dependent, displaying a biphasic behavior as a function of cycle length. Moreover, our results indicate that there is a significant difference in M _S measured between both species at small cycle lengths. Numerical simulations suggest that the kinetics of the rapidly activating delayed rectifier potassium current I _Kr is partially responsible for this difference.     

A Study of Early Afterdepolarizations in a Model for Human Ventricular Tissue

Sudden cardiac death is often caused by cardiac arrhythmias. Recently, special attention has been given to a certain arrhythmogenic condition, the long-QT syndrome, which occurs as a result of genetic mutations or drug toxicity. The underlying mechanisms of arrhythmias, caused by the long-QT syndrome, are not fully understood. However, arrhythmias are often connected to special excitations of cardiac cells, called early afterdepolarizations (EADs), which are depolarizations during the repolarizing phase of the action potential. So far, EADs have been studied mainly in isolated cardiac cells. However, the question on how EADs at the single-cell level can result in fibrillation at the tissue level, especially in human cell models, has not been widely studied yet. In this paper, we study wave patterns that result from single-cell EAD dynamics in a mathematical model for human ventricular cardiac tissue. We induce EADs by modeling experimental conditions which have been shown to evoke EADs at a single-cell level: by an increase of L-type Ca currents and a decrease of the delayed rectifier potassium currents. We show that, at the tissue level and depending on these parameters, three types of abnormal wave patterns emerge. We classify them into two types of spiral fibrillation and one type of oscillatory dynamics. Moreover, we find that the emergent wave patterns can be driven by calcium or sodium currents and we find phase waves in the oscillatory excitation regime. From our simulations we predict that arrhythmias caused by EADs can occur during normal wave propagation and do not require tissue heterogeneities. Experimental verification of our results is possible for experiments at the cell-culture level, where EADs can be induced by an increase of the L-type calcium conductance and by the application of I blockers, and the properties of the emergent patterns can be studied by optical mapping of the voltage and calcium.     

Voltage and Calcium Dynamics Both Underlie Cellular Alternans in Cardiac Myocytes

Cardiac alternans, a putative trigger event for cardiac reentry, is a beat-to-beat alternation in membrane potential and calcium transient. Alternans was originally attributed to instabilities in transmembrane ion channel dynamics (i.e., the voltage mechanism). As of this writing, the predominant view is that instabilities in subcellular calcium handling are the main underlying mechanism. That being said, because the voltage and calcium systems are bidirectionally coupled, theoretical studies have suggested that both mechanisms can contribute. To date, to our knowledge, no experimental evidence of such a dual role within the same cell has been reported. Here, a combined electrophysiological and calcium imaging approach was developed and used to illuminate the contributions of voltage and calcium dynamics to alternans. An experimentally feasible protocol, quantification of subcellular calcium alternans and restitution slope during cycle-length ramping alternans control, was designed and validated. This approach allows simultaneous illumination of the contributions of voltage and calcium-driven instability to total cellular instability as a function of cycle-length. Application of this protocol in in vitro guinea-pig left-ventricular myocytes demonstrated that both voltage- and calcium-driven instabilities underlie alternans, and that the relative contributions of the two systems change as a function of pacing rate.     

Voltage and Calcium Dynamics Both Underlie Cellular Alternans in Cardiac Myocytes

Cardiac alternans, a putative trigger event for cardiac reentry, is a beat-to-beat alternation in membrane potential and calcium transient. Alternans was originally attributed to instabilities in transmembrane ion channel dynamics (i.e., the voltage mechanism). As of this writing, the predominant view is that instabilities in subcellular calcium handling are the main underlying mechanism. That being said, because the voltage and calcium systems are bidirectionally coupled, theoretical studies have suggested that both mechanisms can contribute. To date, to our knowledge, no experimental evidence of such a dual role within the same cell has been reported. Here, a combined electrophysiological and calcium imaging approach was developed and used to illuminate the contributions of voltage and calcium dynamics to alternans. An experimentally feasible protocol, quantification of subcellular calcium alternans and restitution slope during cycle-length ramping alternans control, was designed and validated. This approach allows simultaneous illumination of the contributions of voltage and calcium-driven instability to total cellular instability as a function of cycle-length. Application of this protocol in in vitro guinea-pig left-ventricular myocytes demonstrated that both voltage- and calcium-driven instabilities underlie alternans, and that the relative contributions of the two systems change as a function of pacing rate.     

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca²⁺ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.     

Distribution and Function of Cardiac Ryanodine Receptor Clusters in Live Ventricular Myocytes

The cardiac Ca²⁺ release channel (ryanodine receptor, RyR2) plays an essential role in excitation-contraction coupling in cardiac muscle cells. Effective and stable excitation-contraction coupling critically depends not only on the expression of RyR2, but also on its distribution. Despite its importance, little is known about the distribution and organization of RyR2 in living cells. To study the distribution of RyR2 in living cardiomyocytes, we generated a knock-in mouse model expressing a GFP-tagged RyR2 (GFP-RyR2). Confocal imaging of live ventricular myocytes isolated from the GFP-RyR2 mouse heart revealed clusters of GFP-RyR2 organized in rows with a striated pattern. Similar organization of GFP-RyR2 clusters was observed in fixed ventricular myocytes. Immunofluorescence staining with the anti-α-actinin antibody (a z-line marker) showed that nearly all GFP-RyR2 clusters were localized in the z-line zone. There were small regions with dislocated GFP-RyR2 clusters. Interestingly, these same regions also displayed dislocated z-lines. Staining with di-8-ANEPPS revealed that nearly all GFP-RyR2 clusters were co-localized with transverse but not longitudinal tubules, whereas staining with MitoTracker Red showed that GFP-RyR2 clusters were not co-localized with mitochondria in live ventricular myocytes. We also found GFP-RyR2 clusters interspersed between z-lines only at the periphery of live ventricular myocytes. Simultaneous detection of GFP-RyR2 clusters and Ca²⁺ sparks showed that Ca²⁺ sparks originated exclusively from RyR2 clusters. Ca²⁺ sparks from RyR2 clusters induced no detectable changes in mitochondrial Ca²⁺ level. These results reveal, for the first time, the distribution of RyR2 clusters and its functional correlation in living ventricular myocytes.     

Extracellular Allosteric Na Binding to the Na,K-ATPase in Cardiac Myocytes

Whole-cell patch-clamp measurements of the current, I_p, produced by the Na⁺,K⁺-ATPase across the plasma membrane of rabbit cardiac myocytes show an increase in I_p over the extracellular Na⁺ concentration range 0–50 mM. This is not predicted by the classical Albers-Post scheme of the Na⁺,K⁺-ATPase mechanism, where extracellular Na⁺ should act as a competitive inhibitor of extracellular K⁺ binding, which is necessary for the stimulation of enzyme dephosphorylation and the pumping of K⁺ ions into the cytoplasm. The increase in I_p is consistent with Na⁺ binding to an extracellular allosteric site, independent of the ion transport sites, and an increase in turnover via an acceleration of the rate-determining release of K⁺ to the cytoplasm, E2(K⁺)₂ → E1 + 2K⁺. At normal physiological concentrations of extracellular Na⁺ of 140 mM, it is to be expected that binding of Na⁺ to the allosteric site would be nearly saturated. Its purpose would seem to be simply to optimize the enzyme’s ion pumping rate under its normal physiological conditions. Based on published crystal structures, a possible location of the allosteric site is within a cleft between the α- and β-subunits of the enzyme.     

Mitochondrial Oscillations and Waves in Cardiac Myocytes: Insights from Computational Models

Periodic cellwide depolarizations of mitochondrial membrane potential (Ψ_M) which are triggered by reactive oxygen species (ROS) and propagated by ROS-induced ROS release (RIRR) have been postulated to contribute to cardiac arrhythmogenesis and injury during ischemia/reperfusion. Two different modes of RIRR have been described: Ψ_M oscillations involving ROS-sensitive mitochondrial inner membrane anion channels (IMAC), and slow depolarization waves related to mitochondrial permeability transition pore (MPTP) opening. In this study, we developed a computational model of mitochondria exhibiting both IMAC-mediated RIRR and MPTP-mediated RIRR, diffusively coupled in a spatially extended network, to study the spatiotemporal dynamics of RIRR on Ψ_M. Our major findings are: 1), as the rate of ROS production increases, mitochondria can exhibit either oscillatory dynamics facilitated by IMAC opening, or bistable dynamics facilitated by MPTP opening; 2), in a diffusively-coupled mitochondrial network, the oscillatory dynamics of IMAC-mediated RIRR results in rapidly propagating (∼25 μm/s) cellwide Ψ_M oscillations, whereas the bistable dynamics of MPTP-mediated RIRR results in slow (0.1-2 μm/s) Ψ_M depolarization waves; and 3), the slow velocity of the MPTP-mediated depolarization wave is related to competition between ROS scavenging systems and ROS diffusion. Our observations provide mechanistic insights into the spatiotemporal dynamics underlying RIRR-induced Ψ_M oscillations and waves observed experimentally in cardiac myocytes.     

过氧化氢对培养心肌细胞损伤作用的研究

氧化应激时产生大量的自由基,造成心肌细胞的损伤.过氧化氢(H₂O₂)是有机体氧化代谢产物,同时是一种活性氧.应用不同浓度的H₂O₂,分别于不同作用时间,动态观察其对心肌细胞的损伤作用.从实验结果看到,低浓度的H₂O₂（＜0.1 mmol/L）作用2 h,使心肌细胞产生早期的生物化学的改变,如MDA产生堆积和细胞周期时相改变（G1期细胞增加,G2期细胞减少）,此时心肌酶基本无泄漏,心肌细胞的死亡率很低,HE形态学观察基本无改变;随着H₂O₂浓度的增加（1～5 mmol/L）和作用时间的延长,进一步诱导细胞损伤加剧,LDH释放和MDA积累明显升高,细胞死亡率也明显增加,已具有统计学意义.同时可观察到其病理形态学的坏死性改变;当10 mmol/L H₂O₂作用时,细胞大量死亡,形态学可见细胞极度收缩、脱落,形成大面积的细胞脱失区.因此,H₂O₂作为一种活性氧自由基,依其浓度和作用时间不同可造成不同程度的心肌细胞的损伤.辣根过氧化物酶作为一种自由基清除剂,可明显减少H₂O₂活性氧自由基对心肌细胞的损伤作用.     

Angiotensin II Type 1 Receptor-Mediated Electrical Remodeling in Mouse Cardiac Myocytes

We recently characterized an autocrine renin angiotensin system (RAS) in canine heart. Activation of Angiotensin II Type 1 Receptors (AT₁Rs) induced electrical remodeling, including inhibition of the transient outward potassium current I_to, prolongation of the action potential (AP), increased calcium entry and increased contractility. Electrical properties of the mouse heart are very different from those of dog heart, but if a similar system existed in mouse, it could be uniquely studied through genetic manipulations. To investigate the presence of a RAS in mouse, we measured APs and I_to in isolated myocytes. Application of angiotensin II (A2) for 2 or more hours reduced I_to magnitude, without affecting voltage dependence, and prolonged APs in a dose-dependent manner. Based on dose-inhibition curves, the fast and slow components of I_to (I_to,fast and I_K,slow) appeared to be coherently regulated by [A2], with 50% inhibition at an A2 concentration of about 400 nM. This very high K_0.5 is inconsistent with systemic A2 effects, but is consistent with an autocrine RAS in mouse heart. Pre-application of the microtubule destabilizing agent colchicine eliminated A2 effects on I_to and AP duration, suggesting these effects depend on intracellular trafficking. Application of the biased agonist SII ([Sar¹-Ile⁴-Ile⁸]A2), which stimulates receptor internalization without G protein activation, caused I_to reduction and AP prolongation similar to A2-induced changes. These data demonstrate AT₁R mediated regulation of I_to in mouse heart. Moreover, all measured properties parallel those measured in dog heart, suggesting an autocrine RAS may be a fundamental feedback system that is present across species.