Rat Cardiac Ventricular Myocytes Research Articles

Leonurine pretreatment protects the heart from myocardial ischemia-reperfusion injury.

Myocardial ischemia-reperfusion (I/R), an important complication of reperfusion therapy for myocardial infarction, is characterized by hyperactive oxidative stress and inflammatory response. Leonurine (4-guanidino-n-butyl syringate, SCM-198), an alkaloid extracted from Herbaleonuri, was previously found to be highly cardioprotective both in vitro and in vivo. Our current study aimed to investigate the effect of SCM-198 preconditioning on myocardial I/R injury in vitro and in vivo, respectively, as well as to decipher the mechanism involved. Rats were pretreated with SCM-198 before subjected to 45 min of myocardial ischemia, which was followed by 24 h of reperfusion. Primary neonatal rat cardiac ventricular myocytes (NRCMs) were exposed to hypoxia (95% N2 + 5% CO2) for 12 h, and then to 12 h reoxygenation so as to mimic I/R. The enzymatic measurements demonstrated that SCM-198 reduced the release of infarction-related enzymes, and the hemodynamic and echocardiography measurements showed that SCM-198 restored cardiac functions, which suggested that SCM-198 could significantly reduce infarct size, maintaining cardiomyocyte morphology, and that SCM-198 pretreatment could significantly reduce cardiomyocytes apoptosis. Moreover, we demonstrated that SCM-198 could exert a cardioprotective effect by reducing reactive oxygen species (ROS) level and Akt phosphorylation while reducing the phosphorylation of p38 and JNK. In addition, the upregulation of p-Akt, Bcl-2/Bax induced by SCM-198 treatment were blocked by PI3K inhibitor LY294002, and the total protein level of Akt was not affected by SCM-198 pretreatment. Our experimental results indicated that SCM-198 could have a cardioprotective effect on I/R injury, which confirmed the utility of SCM-198 preconditioning as a strategy to prevent I/R injury.

Protective effects of 18β-Glycyrrhetinic acid against myocardial infarction: Involvement of PI3K/Akt pathway activation and inhibiting Ca2+ influx via L-type Ca2+ channels.

18β‐Glycyrrhetinic acid (18β‐GA) is a component extracted from licorice. This study aimed to evaluate the effects of 18β‐GA on isoproterenol (ISO)‐induced acute myocardial infarction in rats and mice. Two consecutive days of subcutaneous injection of ISO (85 mg/kg/day) resulted in acute myocardial infarction. We examined the pathological changes, oxidative stress, inflammatory response, and expression of apoptosis in mouse hearts. The expressions of phosphoinositol‐3‐kinase (PI3K), protein kinase B (Akt), and the phosphorylation levels of PI3K (p‐PI3K) and Akt (p‐Akt) were determined by western blotting. The whole‐cell patch‐clamp technique was applied to observe the L‐type Ca2+ currents, and the Ion Optix detection system was used for cell contraction and Ca2+ transient in isolated rat cardiac ventricular myocytes. In ISO‐induced myocardial infarction, the J‐point, heart rate, creatine kinase, lactate dehydrogenase, superoxide dismutase, catalase, malondialdehyde, glutathion, and reactive oxygen species decreased in mice after 18β‐GA treatment. 18β‐GA improved ISO‐induced morphologic pathology, inhibited the inflammatory pathway response and cardiomyocyte apoptosis, and inhibited PI3K/Akt signaling. 18β‐GA could significantly inhibit ICa‐L, myocardial contraction, and Ca2+ transient. This study demonstrates that 18β‐GA has cardioprotective effects on acute myocardial infarction, which may be related to inhibiting oxidative stress, inflammation, apoptosis via the PI3K/Akt pathway, and reducing cell contractility and Ca2+ concentration via L‐type Ca2+ channels.

Accounting for cardiac t-tubule increase with age and myocyte volume to improve measurements of its membrane area and ionic current densities

In-silico models of cardiac myocytes allow simulating experiments in numbers on series of myocytes as well as on large populations of myocytes assembled in 3D structures. The simulated myocyte populations should have realistic values and statistical dispersions of biophysical parameters such as myocyte dimensions and volume and areas of the peripheral membrane and transverse-axial tubular system (TATS). Dependencies among these variables also have to be taken into account. In this work, we propose a quantitative representation of the changes in the fraction of membrane area in the TATS that integrates published dependencies with body weight (age) and size of rat ventricular cardiac myocytes while respecting the above constraints. Imposing a constant total membrane area-to-volume ratio appears to account for the increase of this fraction with myocyte size (i.e.: volume) within a given age group. The agreement of our results with published data was discussed and reasons for discrepancies were analysed. On the basis of our framework, strategies are proposed for minimizing the influence of non-random dispersion related to myocyte volume on measurements of the area of TATS and surface membrane compartments and of ionic current densities. The next step will be to quantitatively compare these strategies by evaluating the impact of myocyte morphological parameters and their dependencies, sample size, biases and errors, on the output of experiments.

A0876 MiR-21-3p is related to Angiotensin II-induced hypertrophy in cultured neonatal rat ventricular cardiac myocytes

A0876 MiR-21-3p is related to Angiotensin II-induced hypertrophy in cultured neonatal rat ventricular cardiac myocytes

Sulfur dioxide attenuates sepsis-induced cardiac dysfunction via inhibition of NLRP3 inflammasome activation in rats

ObjectiveSulfur dioxide (SO2) plays an important role in maintaining homeostasis of cardiovascular system. This study was aimed to investigate cardioprotective effects of SO2 on in the rat and the underlying mechanism. Methods and resultsSepsis model induced by cecal ligation and puncture (CLP) in rats were used. SO2 donor (NaHSO3/Na2SO3, 1:3 M/M) was administered intraperitoneally at a dose of 85 mg/kg. Primary neonatal rat cardiac ventricular myocytes (NRCMs) were stimulated with LPS (1 mg/mL) in presence or absence of different concentrations of SO2 (10, 50 and 100 μmol/L). SO2 donor could restore the decreased levels of SO2 in plasma and heart of septic rats. SO2 exhibited dramatic improvement in cardiac functions. At 24 h after CLP, SO2 treatments decreased the number of TUNEL-positive cells, Bax/Bcl-2 ratio and activity of caspase-3. Moreover CLP-induced inflammatory response was also relieved by SO2. In NRCMs, SO2 could suppress the LPS-induced myocardial injury, leading to an increase in cell viability, a decrease in LDH and apoptotic rate. Western blot showed that the expression of TLR4, NLRP3, and Caspase-1 were obviously increased in myocardial tissue of CLP group or in NRCMs of LPS group, while SO2 significantly inhibited the CLP-induced or LPS-induced TLR4, NLRP3, and Caspase-1 expression. ConclusionSO2 attenuated sepsis-induced cardiac dysfunction likely in association with the inhibiting inflammation via TLR4/NLRP3 signaling pathway.

Kinetics of Mechanical Stretch-Induced Nitric Oxide Production in Rat Ventricular Cardiac Myocytes.

Discrete mechanical stretch of isolated spontaneously contracting cardiac myocytes was employed to examine the kinetics of NO production in these cells. NO oscillations were detected with fluorescent dye 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. The mechanisms underlying stretch-induced changes in NO concentration remain unclear and further studies are needed to evaluate the role of NO oscillation in the regulation of cardiomyocyte function.

G protein-coupled receptor kinase 2 promotes cardiac hypertrophy.

The increase in protein activity and upregulation of G-protein coupled receptor kinase 2 (GRK2) is a hallmark of cardiac stress and heart failure. Inhibition of GRK2 improved cardiac function and survival and diminished cardiac remodeling in various animal heart failure models. The aim of the present study was to investigate the effects of GRK2 on cardiac hypertrophy and dissect potential molecular mechanisms. In mice we observed increased GRK2 mRNA and protein levels following transverse aortic constriction (TAC). Conditional GRK2 knockout mice showed attenuated hypertrophic response with preserved ventricular geometry 6 weeks after TAC operation compared to wild-type animals. In isolated neonatal rat ventricular cardiac myocytes stimulation with angiotensin II and phenylephrine enhanced GRK2 expression leading to enhanced signaling via protein kinase B (PKB or Akt), consecutively inhibiting glycogen synthase kinase 3 beta (GSK3β), such promoting nuclear accumulation and activation of nuclear factor of activated T-cells (NFAT). Cardiac myocyte hypertrophy induced by in vitro GRK2 overexpression increased the cytosolic interaction of GRK2 and phosphoinositide 3-kinase γ (PI3Kγ). Moreover, inhibition of PI3Kγ as well as GRK2 knock down prevented Akt activation resulting in halted NFAT activity and reduced cardiac myocyte hypertrophy. Our data show that enhanced GRK2 expression triggers cardiac hypertrophy by GRK2-PI3Kγ mediated Akt phosphorylation and subsequent inactivation of GSK3β, resulting in enhanced NFAT activity.

Mitochondrial Permeability Transition Pore and Number of Openings

Mitochondria are double membrane intracellular organelles that provide the ATP to meet the energy demands of the cell. Mitochondrial dysfunction has been implicated in diverse disease including neurodegenerative pathologies, heart diseases and many more. In rat cardiac ventricular myocytes, due to the large energy demand of a beating heart, mitochondria comprise 30-40% of cell volume and number approximately 20,000 per cell. Within each mitochondrion, the inner mitochondrial membrane (IMM) provide a base for electron transport chain (ETC) and ATP Synthase proteins. The ETC establishes an electrical and pH gradient with the matrix potential of approximately −180 mV. However, depolarizations of the IMM can occur following elevations of reactive oxygen species (ROS) and/or elevations of matrix [Ca2+]m and under other conditions. These depolarizations are largely attributed to the opening of a putative mitochondrial permeability transition pore (mPTP). The exact molecular composition of the mPTP is controversial and over the years has been attributed to many inner and outer membrane protein components as well as other proteins. Recently, the ATP synthase dimers have been implicated as key components of the mPTP as well. Contributions of mPTP to the regulation of matrix Ca2+, mitochondrial volume and programmed cell death (apoptosis) have been suggested. The model suggests that in order to simulate experimental data the ion fluxes that pass through mPTP are mainly Na+ and K+ as these are the most abundant ions present. Furthermore, it takes the opening of only one mPTP for a transient depolarization or sustained depolarization with a current of 30 pA and 26 pA respectively. Constraints on the protein identities and their behaviors are suggested by these models. Importantly, this work suggests that specific experiments be carried out to test candidate proteins and protein-combinations for possible involvement in mPTP.

Novel protective effects of pulsed electromagnetic field ischemia/reperfusion injury rats.

Extracorporeal pulsed electromagnetic field (PEMF) has shown the ability to regenerate tissue by promoting cell proliferation. In the present study, we investigated for the first time whether PEMF treatment could improve the myocardial ischaemia/reperfusion (I/R) injury and uncovered its underlying mechanisms.In our study, we demonstrated for the first time that extracorporeal PEMF has a novel effect on myocardial I/R injury. The number and function of circulating endothelial progenitor cells (EPCs) were increased in PEMF treating rats. The invivo results showed that per-treatment of PEMF could significantly improve the cardiac function in I/R injury group. In addition, PEMF treatment also reduced the apoptosis of myocardial cells by up-regulating the expression of anti-apoptosis protein B-cell lymphoma 2 (Bcl-2) and down-regulating the expression of pro-apoptosis protein (Bax). Invitro, the results showed that PEMF treatment could significantly reduce the apoptosis and reactive oxygen species (ROS) levels in primary neonatal rat cardiac ventricular myocytes (NRCMs) induced by hypoxia/reoxygenation (H/R). In particular, PEMF increased the phosphorylation of protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS), which might be closely related to attenuated cell apoptosis by increasing the releasing of nitric oxide (NO). Therefore, our data indicated that PEMF could be a potential candidate for I/R injury.

Isolated neonatal rat papillary muscles: a new model to translate neonatal rat myocyte signaling into contractile mechanics.

Isolated cardiac tissue allows investigators to study mechanisms underlying normal and pathological conditions, which would otherwise be difficult or impossible to perform invivo. Cultured neonatal rat ventricular cardiac myocytes (NRVM) are widely used to study signaling and growth mechanisms in the heart, primarily due to the versatility, economy, and convenience of this invitro model. However, the lack of a well-defined longitudinal cellular axis greatly hampers the ability to measure contractile function in these cells, and therefore to associate signaling with mechanical function. In these methods, we demonstrate that this limitation can be overcome by using papillary muscles isolated from neonatal rat hearts. In the methods we describe procedures for isolation of right ventricular papillary muscles from 3-day-old neonatal rats and effects of mechanical and humoral stimuli on contraction and relaxation properties of these tissues.

Detachment of surface membrane invagination systems by cationic amphiphilic drugs.

Several cell types develop extensive plasma membrane invaginations to serve a specific physiological function. For example, the megakaryocyte demarcation membrane system (DMS) provides a membrane reserve for platelet production and muscle transverse (T) tubules facilitate excitation:contraction coupling. Using impermeant fluorescent indicators, capacitance measurements and electron microscopy, we show that multiple cationic amphiphilic drugs (CADs) cause complete separation of the DMS from the surface membrane in rat megakaryocytes. This includes the calmodulin inhibitor W-7, the phospholipase-C inhibitor U73122, and anti-psychotic phenothiazines. CADs also caused loss of T tubules in rat cardiac ventricular myocytes and the open canalicular system of human platelets. Anionic amphiphiles, U73343 (a less electrophilic U73122 analogue) and a range of kinase inhibitors were without effect on the DMS. CADs are known to accumulate in the inner leaflet of the cell membrane where they bind to anionic lipids, especially PI(4,5)P2. We therefore propose that surface detachment of membrane invaginations results from an ability of CADs to interfere with PI(4,5)P2 interactions with cytoskeletal or BAR domain proteins. This establishes a detubulating action of a large class of pharmaceutical compounds.

PP2A Level in Colorectal Cancer Cells Predicts the Response of p38 Targeted Therapy

PP2A Level in Colorectal Cancer Cells Predicts the Response of p38 Targeted Therapy

PI4P Hydrolysis Represents a General Mechanism for DAG Generation and PKC/PKD Activation

Receptor stimulated hydrolysis of phosphatidylinositol 4,5‐bisphosphate (PIP­2­) is critical for many physiological processes, such as neurotransmission and proliferation. Recently our laboratory identified a novel pathway in neonatal rat ventricular cardiac myocytes (NRVMs), where agonist stimulation triggers PLC dependent hydrolysis of the PIP­2­ precursor phosphatidylinositol 4‐phosphate (PI4P) leading to DAG production, nuclear PKD activation and cardiac hypertrophy. We have seen that this newly identified pathway exists in other cell types as well, such as Mouse Embryo Fibroblasts (MEFs) and PANC‐1 cells. To determine if PLC dependent PI4P hydrolysis represents a general mechanism for DAG generation and PKC/PKD activation we examined PI4P depletion and PKD activation in other cell types. In our studies we used a PI 4‐kinase inhibitor, phenylarsine oxide (PAO) or expression of a Golgi specific 4‐phosphatase, Sac1‐K2A (Sac1) to deplete PI4P. PAO treatment or Sac1 expression in NRVMs and MEFs, showed a dramatic reduction of inositol phosphate accumulation after endothelin‐1 (ET‐1) stimulation. PAO treatment completely blocked ET‐1 dependent global PKD activation in NRVMs and MEFs but not neurotensin (NT) dependent PKD activation in PANC‐1 cells. Sac1 expression showed reduced ET‐1 dependent global PKD activation in NRVMs and MEFs but did not change the NT dependent PKD activation in PANC‐1 cells. These results suggest that though Golgi PI4P hydrolysis occurs in multiple cell types, NRVMs and MEFs use PI4P hydrolysis as a source for DAG to promote PKD activation. This shows that PI4P hydrolysis occurs upon receptor stimulation and provides a novel signaling mechanism with numerous implications in physiology and disease.

The potassium current carried by TREK-1 channels in rat cardiac ventricular muscle.

We studied the potassium current flowing through TREK-1 channels in rat cardiac ventricular myocytes. We separated the TREK-1 current from other current components by blocking most other channels with a blocker cocktail. We tried to inhibit the TREK-1 current by activating protein kinase A (PKA) with a mixture of forskolin and isobutyl-methylxanthine (IBMX). Activation of PKA blocked an outwardly rectifying current component at membrane potentials positive to -40 mV. At 37 °C, application of forskolin plus IBMX reduced the steady-state outward current measured at positive voltages by about 52 %. Application of the potassium channel blockers quinidine or tetrahexylammonium also reduced the steady-state outward current by about 50 %. Taken together, our results suggest that the increase in temperature from 22 to 37 °C increased the TREK-1 current by a factor of at least 5 and that the average density of the TREK-1 current in rat cardiomyocytes at 37 °C is about 1.5 pA/pF at +30 mV. The contribution of TREK-1 to the action potential was assessed by using a dynamic patch clamp technique. After subtraction of simulated TREK-1 currents, action potential duration at 50 or 90 % repolarisation was increased by about 12 %, indicating that TREK-1 may be functionally important in rat ventricular muscle. During sympathetic stimulation, inhibition of TREK-1 channels via PKA is expected to prolong the action potential primarily in subendocardial myocytes; this may decrease the transmural dispersion of repolarisation and thus may serve to prevent the occurrence of arrhythmias.

Abstract 16606: Cardiac Fibroblast-Restricted Enhancement of G q Signaling Prolongs Calcium Transients and Increases Spontaneous Activity in Biomimetic Cardiac Microtissues

Introduction: Enhanced G q signaling in response to hemodynamic stress and injury is well known to induce remodeling in both cardiac myocytes (CMs) and fibroblasts (CFs). However, the effect of CF activation and fibrosis on CMs and integrated myocardial function cannot be easily discerned from concomitant effects on CMs. Hypothesis: Enhanced G q signaling in CFs influences CM function in a 3D microtissue model previously developed in our lab, in which neonatal rat ventricular CMs and CFs are highly interspersed as seen in the myocardium. Methods: CFs infected in suspension (2 hrs, 10 MOI) with empty adenovirus (Ad-Ctr) or Ad encoding wild-type (WT) or constitutively active (Q209L or QL) Gα q were co-seeded with uninfected CMs (CM:CF 1:1) into non-adhesive hydrogels at ~1200 cells per recess (400/800 μm wide/deep). After 2-4 days, we assessed tissue size by microscopy, G q and extracellular matrix proteins proteins by Western Blot analysis, and Ca 2+ transients ([Ca 2+ ] i ) with Rhod 2/AM. Results: Overexpression of Gα q in CFs was associated with a 3-fold (WT) and 7-fold (QL) increase in total inositolphosphate formation, indicating enhanced basal phospholipase β activity. After 4 days, CM:CF QL tissues were larger in size by 30-40% and expressed more laminin and fibronectin. After 3 days, CM:CF CTR paced at 1 Hz had [Ca 2+ ] i duration of 218±3 ms, decay of 141±2 ms, and rise time of 35±4 ms (n=55-69). In CM:CF QL , duration and decay were prolonged by 160 ms and 21 ms, resp., and rise time was decreased by 5 ms (n=10-27). In CM:CF WT with less G q signaling, [Ca 2+ ] i duration and decay were also prolonged but only by 30 ms and 7 ms, resp. (n=46-51). These effects were similar but less pronounced after 2 days and/or at 2 Hz. Spontaneous [Ca 2+ ] i activity was seen in 50% of CM:CF QL , 11% of CM:CF WT , but only 2% of CM:CF CTR microtissues. The gap junction inhibitor carbenoxolone (100 μM) decreased this activity in CM:CF QL by 80% and in CM:CF WT by 15%. Conclusion: We show that enhanced CF-restricted G q signaling in biomimetic cardiac microtissues increases tissue size, prolongs [Ca 2+ ] i duration and decay, and increases spontaneous Ca 2+ activity. Our findings suggest that depending on the CF activation state, CFs can greatly modulate CM calcium handling and potentially influence arrhythmogenesis.

Compartmentation of Camp Signaling in Complex and Simple Cells

Differences in compartmentation of cAMP signaling were compared in adult rat cardiac ventricular myocytes and HEK293 cells. The freely diffusible Epac2-camps FRET-based biosensor was used to monitor cAMP responses in the bulk cytoplasmic compartment of these cells, while Epac2-MyrPalm and Epac2-CAAX versions of the probe were used to measure subcellular cAMP responses associated with lipid raft and non-lipid raft domains of the plasma membrane, respectively. Stimulating raft associated beta-adrenergic receptors (βAR) or non-raft associated E-type prostaglandin receptors (EPR) elicited markedly different cAMP responses in the two cell types. In HEK293 cells, maximal βAR or EPR stimulation produced saturating cAMP responses in all three domains. In cardiac myocytes, maximal βAR stimulation produced non-saturating responses in all three domains. However, EPR stimulation produced responses that were smaller, transient, and more consistently observed in non-lipid raft and bulk cytoplasmic domains. There were also significant differences in the pattern of basal cAMP activity associated with the different microdomains of the two cell types. Direct inhibition of adenylyl cyclase (AC) activity with MDL12330A (MDL) only produced a decrease in basal cAMP activity in non-lipid raft domains of HEK293 cells. However, MDL inhibited basal cAMP activity in non-lipid raft domains as well in the bulk cytoplasmic compartment of cardiac myocytes. In cardiac myocytes, responses detected in all three locations were significantly more sensitive to inhibition of phosphodiesterase (PDE) activity. However, in HEK293 cells, responses detected in non-lipid raft domains were more sensitive to direct stimulation of AC activity with forskolin. Computational modeling suggests that AC activity may play a more important role in explaining the compartmentalized responses observed in HEK293 cells, while PDE activity may be more important in cardiac myocytes.

Functional subcellular distribution of β1 - and β2 -adrenergic receptors in rat ventricular cardiac myocytes

β-adrenergic stimulation is a key regulator of cardiac function. The localization of major cardiac adrenergic receptors (β1 and β2) has been investigated using biochemical and biophysical approaches and has led to contradictory results. This study investigates the functional subcellular localization of β1- and β2-adrenergic receptors in rat ventricular myocytes using a physiological approach. Ventricular myocytes were isolated from the hearts of rat and detubulated using formamide. Physiological cardiac function was measured as Ca2+ transient using Fura-2-AM and cell shortening. Selective activation of β1- and β2-adrenergic receptors was induced with isoproterenol (0.1 μmol/L) and ICI-118,551 (0.1 μmol/L); and with salbutamol (10 μmol/L) and atenolol (1 μmol/L), respectively. β1- and β2-adrenergic stimulations induced a significant increase in Ca2+ transient amplitude and cell shortening in intact rat ventricular myocytes (i.e., surface sarcolemma and t-tubules) and in detubulated cells (depleted from t-tubules, surface sarcolemma only). Both β1- and β2-adrenergic receptors stimulation caused a greater effect on Ca2+ transient and cell shortening in detubulated myocytes than in control myocytes. Quantitative analysis indicates that β1-adrenergic stimulation is ∼3 times more effective at surface sarcolemma compared to t-tubules, whereas β2- adrenergic stimulation occurs almost exclusively at surface sarcolemma (∼100 times more effective). These physiological data demonstrate that in rat ventricular myocytes, β1-adrenergic receptors are functionally present at surface sarcolemma and t-tubules, while β2-adrenergic receptors stimulation occurs only at surface sarcolemma of cardiac cells.

42. Investigation into cardiomyocyte response to cryoablation

Atrial and ventricular tachyarrhythmias represent major cardiovascular diseases that may be treated utilizing thermal therapies. Given this there has been a palpable increase in technologies and approaches of therapeutic intervention, including cryoablation. While increased utilization of cryoablation continues, our fundamental understanding of the response of cardiac cells to the freezing process remains limited. As such we investigated the cellular and molecular response of cardiomyocytes to a range of subfreezing temperatures in an effort to provide further insight to help guide the application of cryoablation for the treatment of cardiac arrhythmias. Primary neonatal rat ventricular cardiac myocytes (NRVCM) and human cardiac myocytes (HCM) were cultured in vitro to study the cellular and molecular effects of thermal excursions. Cells were exposed to a range of temperatures from 37 °C to −40 °C for up to 1 min. Thermal profiles were generated to examine the cooling rates at each target temperature. Samples were then returned to culture and cell survival was measured via metabolic activity, membrane integrity and contractile function. Microfluidic flow cytometry was also conducted to investigate apoptotic involvement and confirm viability assessments of the various populations in a time-course manner. Exposure of NRVCM to temperatures ranging from 37 °C to 0 °C resulted in a minimal reduction in cell viability. Exposure to −5 °C yielded 50% cell death. Temperatures ranging from −10 °C to −20 °C yielded less than 20% survival, whereas exposure to −40 °C resulted in complete cell death. Regardless of the level of cell survival, exposure to temperatures below −5 °C resulted in a loss of spontaneous contraction in surviving cells. HCM demonstrated moderate sensitivity (<30% loss) to temperatures above −10 °C, however a steep decline in cell survival was observed from −12 °C to −25 °C, the point at which all cells were metabolically inactive. Flow cytometric analysis revealed the activation of apoptosis in cardiomyocytes in response to the freezing insult. The level and timing was found to vary with temperature, but not duration of the freeze. Although the utilization of cryoablation for the treatment of arrhythmias is established clinically, there remains a void in our knowledge base of the cellular and molecular responses of cardiac systems to thermal stress. This study, as well as others, are now providing mechanistic answers to the how cryoablation works at the cellular level as well as providing critical characterization data to help guide application and future development of therapeutic application.

Wenxin Keli attenuates ischemia-induced ventricular arrhythmias in rats: Involvement of L-type calcium and transient outward potassium currents

Wenxin Keli is the first state‑sanctioned traditional Chinese medicine (TCM)-based antiarrhythmic drug. The present study aimed to examine whether long‑term treatment with Wenxin Keli reduces ischemia‑induced ventricular arrhythmias in rats in vivo, and if so, which mechanisms are involved. Male rats were treated with either saline (control group) or Wenxin Keli for 3 weeks and were subjected to myocardial ischemia for 30 min with assessment of the resulting ventricular arrhythmias. The L‑type calcium current (ICa,L) and transient outward potassium current (Ito) were measured by the patch clamp technique in normal rat cardiac ventricular myocytes. During the 30‑min ischemia, Wenxin Keli significantly reduced the incidence of ventricular fibrillation (VF) (P<0.05). The number of ventricular tachycardia (VT)+VF episodes and the severity of arrhythmias were significantly reduced by Wenxin Keli administration compared to the control group (P<0.05). In addition, Wenxin Keli inhibited ICa,L and Ito in a concentration‑dependent manner. These results suggest that long‑term treatment with Wenxin Keli may attenuate ischemia‑induced ventricular arrhythmias in rats and that ICa,L and Ito may be involved in this attenuation.

D2 dopamine receptor antagonist raclopride induces non‐canonical autophagy in cardiac myocytes

Cell death by autophagy is an important means of maintaining cellular homeostasis in adult cardiac myocytes. Autophagy was previously shown to exert a cardioprotective effect, suggesting that modulation of autophagy pathways is a potential therapeutic strategy in the treatment of heart disease. Although dopamine is known to induce autophagy in neuroblastoma cells, the underlying mechanism and the types of dopamine receptors involved in this process remain unclear. In this study, we used various dopamine receptor antagonists and agonists to identify the specific dopamine receptor that mediates induction of autophagy. We evaluated autophagy induction by the expression of autophagy markers in neonatal rat ventricular cardiac myocytes. We evaluated intracellular calcium levels using Fluo-3/AM and demonstrated autophagy-induced morphological changes in cardiac myocytes using electron microscopy. We also examined the pathway for dopamine-induced autophagy using RNAi-mediated gene knockdown. Raclopride, the well-documented D2 receptor antagonist, significantly upregulated autophagy in cardiac myocytes via an mTOR-independent pathway. There was no difference in intracellular calcium levels between raclopride-treated cells and untreated cells. siRNA-mediated knockdown of Rab9 resulted in decreased expression of autophagy markers in raclopride-treated cells. Interestingly, siRNA-mediated knockdown of Atg7 resulted in a significant increase in Rab9 levels in raclopride-treated cells, suggesting that blocking the classical autophagy pathway results in activation of an alternative pathway. Our study suggests that (1) the D2 dopamine receptor plays a role in autophagy and (2) raclopride mediated a non-canonical autophagy pathway in cardiac myocytes via Rab9.