Video-based Edge Detection Research Articles

Mono- and Biallelic Protein-Truncating Variants in Alpha-Actinin 2 Cause Cardiomyopathy Through Distinct Mechanisms.

ACTN2 (alpha-actinin 2) anchors actin within cardiac sarcomeres. The mechanisms linking ACTN2 mutations to myocardial disease phenotypes are unknown. Here, we characterize patients with novel ACTN2 mutations to reveal insights into the physiological function of ACTN2. Patients harboring ACTN2 protein-truncating variants were identified using a custom mutation pipeline. In patient-derived iPSC-cardiomyocytes, we investigated transcriptional profiles using RNA sequencing, contractile properties using video-based edge detection, and cellular hypertrophy using immunohistochemistry. Structural changes were analyzed through electron microscopy. For mechanistic studies, we used co-immunoprecipitation for ACTN2, followed by mass-spectrometry to investigate protein-protein interaction, and protein tagging followed by confocal microscopy to investigate introduction of truncated ACTN2 into the sarcomeres. Patient-derived iPSC-cardiomyocytes were hypertrophic, displayed sarcomeric structural disarray, impaired contractility, and aberrant Ca2+-signaling. In heterozygous indel cells, the truncated protein incorporates into cardiac sarcomeres, leading to aberrant Z-disc ultrastructure. In homozygous stop-gain cells, affinity-purification mass-spectrometry reveals an intricate ACTN2 interactome with sarcomere and sarcolemma-associated proteins. Loss of the C-terminus of ACTN2 disrupts interaction with ACTN1 (alpha-actinin 1) and GJA1 (gap junction protein alpha 1), 2 sarcolemma-associated proteins, which may contribute to the clinical arrhythmic and relaxation defects. The causality of the stop-gain mutation was verified using CRISPR-Cas9 gene editing. Together, these data advance our understanding of the role of ACTN2 in the human heart and establish recessive inheritance of ACTN2 truncation as causative of disease.

Activation of TRPC (Transient Receptor Potential Canonical) Channel Currents in Iron Overloaded Cardiac Myocytes.

Arrhythmias and heart failure are common cardiac complications leading to substantial morbidity and mortality in patients with hemochromatosis, yet mechanistic insights remain incomplete. We investigated the effects of iron (Fe) on electrophysiological properties and intracellular Ca2+ (Ca2+i) handling in mouse left ventricular cardiomyocytes. Cardiomyocytes were isolated from the left ventricle of mouse hearts and were superfused with Fe3+/8-hydroxyquinoline complex (5-100 μM). Membrane potential and ionic currents including TRPC (transient receptor potential canonical) were recorded using the patch-clamp technique. Ca2+i was evaluated by using Fluo-4. Cell contraction was measured with a video-based edge detection system. The role of TRPCs in the genesis of arrhythmias was also investigated by using a mathematical model of a mouse ventricular myocyte with the incorporation of the TRPC component. We observed prolongation of the action potential duration and induction of early and delayed afterdepolarizations in myocytes superfused with 15 µmol/L Fe3+/8-hydroxyquinoline complex. Iron treatment decreased the peak amplitude of the L-type Ca2+ current and total K+ current, altered Ca2+i dynamics, and decreased cell contractility. During the final phase of Fe treatment, sustained Ca2+i waves and repolarization failure occurred and ventricular cells became unexcitable. Gadolinium abolished Ca2+i waves and restored the resting membrane potential to the normal range. The involvement of TRPC activation was confirmed by TRPC channel current recordings in the absence or presence of functional TRPC channel antibodies. Computer modeling captured the same action potential and Ca2+i dynamics and provided additional mechanistic insights. We conclude that iron overload induces cardiac dysfunction that is associated with TRPC channel activation and alterations in membrane potential and Ca2+i dynamics.

Abstract 507: Activation of Transient Receptor Potential Canonical Channel Currents in Iron-Overloaded Cardiac Myocytes

Background: Iron (Fe) overload cardiomyopathy is the leading cause of death in hemochromatotic patients, yet the mechanistic insight is still incomplete and controversial. We investigated alterations of action potentials (APs), ionic currents, and intracellular Ca 2+ (Ca 2+ i ) in Fe-loaded mouse cardiomyocytes, as well as functional impacts of Fe overload on single-cell contraction and whole-heart arrhythmias. Methods: Cardiomyocytes were isolated from left ventricles of mouse hearts and were superfused with Fe 3+ /8-hydroxyquinoline complex (5-100 μM). APs, L-type Ca 2+ currents (I Ca,L ), total outward K + currents (I K ), and transient receptor potential canonical (TRPC) channel currents were recorded by the patch-clamp technique. Ca 2+ i was evaluated by using Fluo-4. Cell contraction was measured by a video-based edge detection system. Arrhythmias were evaluated in Langendorff-perfused hearts under S 1 -S 2 stimulation protocol. Results: Persistent Fe (15 μM) treatment prolonged AP duration at 90% repolarization (APD 90 : 46.8 ± 2.8 vs. 203.6 ± 63.4 ms, p<0.05), induced early and delayed afterdepolarizations (EADs: 0 % vs. 45.0 ± 15.0 %; DADs: 4.3 ± 1.4 vs. 27.0 ± 7.0 %, p<0.05, respectively) in mouse cardiomyocytes. Consistently, arrhythmia incidence was increased in Fe 3+ /8-HQ-perfused hearts. Fe treatment decreased peak I Ca,L (16.5 ± 1.7 vs.11.4 ± 1.3 pA/pF, p<0.01) and I K (59.2 ± 3.3 vs. 50.4 ± 3.0 pA/pF, p<0.01), altered Ca 2+ i transient patterns and decreased contractility (4.8 ± 0.5 vs. 3.5 ± 0.4%, p<0.01). During the late phase of Fe treatment, fast Ca 2+ waves and sustained depolarization were induced to generate a secondary (shallow) resting membrane potential (RMP: from -68.8 ± 0.6 to -25.0 ± 3.7 mV) where the myocytes became unexcitable. Gadolinium, a TRPC channel blocker, abolished fast Ca 2+ waves and reversed RMP to the deep level (-62.9 ± 3.5 mV). The involvement of TRPC activation was determined for the first time by recording TRPC current and assessing the effect of functional TRPC channel antibodies. Conclusions: In mouse cardiomyocytes, Fe overload induced arrhythmogenic APD prolongation and EADs/DADs, aberrant Ca 2+ i dynamics, and impaired contractility. The activation of TRPC channels accounts for an important underlying mechanism.

Thyroid Hormone Deficiency Adversely Affects Cardiomyocyte T‐tubule Structure and Function

Thyroid hormone deficiency causes adverse changes in cardiomyocyte shape leading to increased wall stress, chamber dilatation and in the long‐term can result in heart failure. We hypothesized that thyroid hormones regulate cardiac contractile function in part by maintaining T‐tubule (TT) structures and efficient excitation‐contraction (EC)‐coupling.MethodsFemale Sprague‐Dawley rats (aged 12 wks) were surgically thyroidectomized (Tx) or were maintained as euthyroid (Eu) controls (6 rats/group). The study was terminated after 12 mo. at which time echocardiographic and hemodynamic measurements were recorded. Ventricular myocytes were isolated by collagenase digestion and plated onto laminin‐coated glass coverslips for TT analysis or were analyzed immediately for contractile and calcium dynamic using videobased edge detection of sarcomere length changes coupled to a fluorescence imaging system (IonOptix). Live cells were labeled with wheat germ agglutinin‐coupled to Alexa‐Fluor (WGA‐488) to image the sarcolemma and TT by confocal microscopy. Multiple z‐stack images were captured per cell and TT periodicity was analyzed using an algorithm to calculate periodicity based on the peak amplitude in the Fourier spectrum of the image at the TT frequency, or TT power. This software program is available as TTorg plugin on ImageJ.ResultsEchocardiography and in vivo hemodynamic measurements showed significantly reduced systolic and diastolic function in Tx vs euthyroid rats. In response to 1 Hz field stimulation at 4V, isolated cardiomyocyte contractile cycles showed significant dysfunction in Tx vs Eu cardiomyocytes: slower max contraction velocity (+Vmax), −2.047±0.30 vs −3.503±0.50 um/sec, Tx vs Eu, respectively, p<0.001; slower max relaxation velocity (‐Vmax), 1.369±0.27 vs 2.44±0.73 um/sec, p<0.01; longer time to peak contraction, 0.279±0.03 vs 0.182±0.03 sec, p<0.001. Myocytes were loaded with Fura2/AM (1uM) and intracellular calcium measured by fluorescence intensity ratio at 340/380 nm. Ca2+ transients showed slower kinetics in Tx vs Eu cardiomyocytes: time to Ca2+ peak, 0.076±0.01 vs 0.0516±0.003 sec, Tx vs Eu, respectively, p<0.0001; time to Ca2+ max return velocity, 0.261 0.03 vs 0.184 0.005, Tx vs Eu, respectively, p<0.0001. TTorg analysis indicated significant disorganization of T‐tubules in Tx cardiomyocytes compared to Eu (TT power = 61±2 vs 80±2, Tx vs Eu, respectively, p<0.00001; arbitrary units with higher values indicate increased periodicity). In conclusion, TH‐deficiency disrupts T‐tubule structures causing cardiomyocyte contractile dysfunction and slower calcium kinetics indicating altered EC‐coupling. Therefore maintaining normal thyroid function is necessary for optimal heart health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Magnesium isoglycyrrhizinate inhibits L-type Ca2+ channels, Ca2+ transients, and contractility but not hERG K+ channels.

To explore the cardiovascular protective effects of Magnesium isoglycyrrhizinate (MI), especially the underlying cellular mechanisms related to L-type calcium channels and myocardial contractility, and to examine the effects of MI on hERG K+ current expressed in HEK293 cells. We used the whole-cell patch clamp technique, video-based edge detection and dual excitation fluorescence photomultiplier systems to explore the effect of MI on L-type Ca2+ currents (ICa-L) and cell contraction in rat cardiomyocytes. We also examined the rapidly activating delayed rectifier potassium current (IKr) expressed in HEK293 cells using a perforated patch clamp. MI inhibited ICa-L in a dose-dependent manner, with a half-maximal inhibitory concentration (IC50) of 0.22mg/ml, and the maximal inhibitory effect was 61.10±0.59%. MI at a concentration of 0.3mg/ml reduced cell shortening by 24.12±3.97% and the peak value of the Ca2+ transient by 36.54±4.96%. MI had no significant influence on hERG K+ channels expressed in HEK293 cells at all test potentials. MI exerts protective effects on the heart via the inhibition of ICa-L and cell shortening in rat cardiomyocytes. However, MI had no significant influence on IKr; thus, MI may exert cardioprotective effects without causing drug-induced long QT syndrome.

Bufalin, a bufanolide steroid from the parotoid glands of the Chinese toad, inhibits L-type Ca2+ channels and contractility in rat ventricular myocytes.

Bufalin is a bufanolide steroid compound in Chan Su. Chan Su is a traditional Chinese medicine prepared from the dried white secretion of the auricular and skin glands of toads and has been used as an oriental drug. However, the effect of bufalin on cardiac function and its underlying cellular mechanisms remain unclear. Here, we explore the cellular mechanisms of bufalin on myocardial protection via the whole-cell patch-clamp recording and video-based edge detection system. Exposure to bufalin resulted in a concentration-dependent blockade of ICa-L , with the half-maximal inhibitory concentration (IC50 ) of 60 μm and the maximal inhibitory effect of 71.50 ± 2.67%. Bufalin at 100 μm reduced cell shortening by 33.83 ± 4.01%. Bufalin restrained L-type Ca2+ channels conductance, and contractility in rat ventricular myocytes. Thus, the protective effects of bufalin on the heart may be determined by the inhibitory effect on ICa-L and the negative inotropic action caused by the decrease of intracellular Ca2+ in rat myocardial cells.

Cardiac Non-myocyte Cells Show Enhanced Pharmacological Function Suggestive of Contractile Maturity in Stem Cell Derived Cardiomyocyte Microtissues.

The immature phenotype of stem cell derived cardiomyocytes is a significant barrier to their use in translational medicine and pre-clinical in vitro drug toxicity and pharmacological analysis. Here we have assessed the contribution of non-myocyte cells on the contractile function of co-cultured human embryonic stem cell derived cardiomyocytes (hESC-CMs) in spheroid microtissue format. Microtissues were formed using a scaffold free 96-well cell suspension method from hESC-CM cultured alone (CM microtissues) or in combination with human primary cardiac microvascular endothelial cells and cardiac fibroblasts (CMEF microtissues). Contractility was characterized with fluorescence and video-based edge detection. CMEF microtissues displayed greater Ca2+ transient amplitudes, enhanced spontaneous contraction rate and remarkably enhanced contractile function in response to both positive and negative inotropic drugs, suggesting a more mature contractile phenotype than CM microtissues. In addition, for several drugs the enhanced contractile response was not apparent when endothelial cell or fibroblasts from a non-cardiac tissue were used as the ancillary cells. Further evidence of maturity for CMEF microtissues was shown with increased expression of genes that encode proteins critical in cardiac Ca2+ handling (S100A1), sarcomere assembly (telethonin/TCAP) and β-adrenergic receptor signalling. Our data shows that compared with single cell-type cardiomyocyte in vitro models, CMEF microtissues are superior at predicting the inotropic effects of drugs, demonstrating the critical contribution of cardiac non-myocyte cells in mediating functional cardiotoxicity.

New Findings on the Effects of Tannic Acid: Inhibition of L-Type Calcium Channels, Calcium Transient and Contractility in Rat Ventricular Myocytes.

Tannic acid (TA) is a group of water-soluble polyphenolic compounds that occur mainly in plant-derived feeds, food grains and fruits. Many studies have explored its biomedical properties, such as anticancer, antibacterial, antimutagenic, antioxidant, antidiabetic, antiinflammatory and antihypertensive activities. However, the effects of TA on the L-type Ca(2+) current (ICa-L) of cardiomyocytes remain undefined. The present study examined the effects of TA on ICa-L using the whole-cell patch-clamp technique and on intracellular Ca(2+) handling and cell contractility in rat ventricular myocytes with the aid of a video-based edge detection system. Exposure to TA resulted in a concentration- and voltage-dependent blockade of ICa-L, with the half maximal inhibitory concentration of 1.69 μM and the maximal inhibitory effect of 46.15%. Moreover, TA significantly inhibited the amplitude of myocyte shortening and peak value of Ca(2+) transient and increased the time to 10% of the peak. These findings provide new experimental evidence for the cellular mechanism of action of TA and may help to expand clinical treatments for cardiovascular disease.

Matrigel Mattress: A Method for the Generation of Single Contracting Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

The lack of measurable single-cell contractility of human-induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery. To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult CMs. Single hiPSC-CMs were cultured for 5 to 7 days on a 0.4- to 0.8-mm thick mattress of undiluted Matrigel (mattress hiPSC-CMs) and compared with hiPSC-CMs maintained on a control substrate (<0.1-mm thick 1:60 diluted Matrigel, control hiPSC-CMs). Compared with control hiPSC-CMs, mattress hiPSC-CMs had more rod-shape morphology and significantly increased sarcomere length. Contractile parameters of mattress hiPSC-CMs measured with video-based edge detection were comparable with those of freshly isolated adult rabbit ventricular CMs. Morphological and contractile properties of mattress hiPSC-CMs were consistent across cryopreserved hiPSC-CMs generated independently at another institution. Unlike control hiPSC-CMs, mattress hiPSC-CMs display robust contractile responses to positive inotropic agents, such as myofilament calcium sensitizers. Mattress hiPSC-CMs exhibit molecular changes that include increased expression of the maturation marker cardiac troponin I and significantly increased action potential upstroke velocity because of a 2-fold increase in sodium current (INa). The Matrigel mattress method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single-cell level, which should be valuable to disease modeling, drug discovery, and preclinical cardiotoxicity testing.

Abstract 18251: Establishment of an in vitro Platform, the Hydrogel Mattress, to Enhance Maturation and Evaluate Contractile Function of Individual hiPSC-CMs

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have great potential as tools for cell based therapy, disease modeling and drug discovery. However, their contractile properties have not been routinely evaluated; as methods to do so are cumbersome and not easily translated to standard laboratories. We sought to develop a more efficient protocol to evaluate hiPSC-CM mechanical properties, at the single cell level. Individual hiPSC-CMs were cultured on a hydrogel based platform, termed the hydrogel mattress, and their cellular contractile properties were evaluated using video based edge detection (Figure A). We found individual hiPSC-CMs maintained for 5 days on the mattress assumed an elongated shape (Figure B) and exhibited robust, reproducible cell shortening (~10% versus &lt;1% for standard culture). We further found hiPSC-CM cellular contraction and peak cell shortening amplitude was comparable to that of freshly isolated adult rabbit and mouse ventricular CMs (Figure C). In addition, hiPSC-CMs maintained on the mattress exhibited characteristics of mature CMs including action potential waveform, calcium handling and pharmacological response to myofilament calcium sensitizers. We further show a positive correlation with the established traction force microscopy method. We demonstrate the hydrogel mattress platform enables routine characterization and quantification of contractile performance of isolated hiPSC-CMs. This platform can be extended to in vitro disease modeling, drug discovery and drug induced cardiotoxicity testing. Further, the mattress can be easily adapted to virtually any laboratory, thus streamlining cellular mechanical evaluation of individual hiPSC-CMs in vitro.

Ketamine attenuates the Na+-dependent Ca2+ overload in rabbit ventricular myocytes in vitro by inhibiting late Na+ and L-type Ca2+ currents.

Intracellular Ca(2+) ([Ca(2+)]i) overload occurs in myocardial ischemia. An increase in the late sodium current (INaL) causes intracellular Na(+) overload and subsequently [Ca(2+)]i overload via the reverse-mode sodium-calcium exchanger (NCX). Thus, inhibition of INaL is a potential therapeutic target for cardiac diseases associated with [Ca(2+)]i overload. The aim of this study was to investigate the effects of ketamine on Na(+)-dependent Ca(2+) overload in ventricular myocytes in vitro. Ventricular myocytes were enzymatically isolated from hearts of rabbits. INaL, NCX current (INCX) and L-type Ca(2+) current (ICaL) were recorded using whole-cell patch-clamp technique. Myocyte shortening and [Ca(2+)]i transients were measured simultaneously using a video-based edge detection and dual excitation fluorescence photomultiplier system. Ketamine (20, 40, 80 μmol/L) inhibited INaL in a concentration-dependent manner. In the presence of sea anemone toxin II (ATX, 30 nmol/L), INaL was augmented by more than 3-fold, while ketamine concentration-dependently suppressed the ATX-augmented INaL. Ketamine (40 μmol/L) also significantly suppressed hypoxia or H2O2-induced enhancement of INaL. Furthermore, ketamine concentration-dependently attenuated ATX-induced enhancement of reverse-mode INCX. In addition, ketamine (40 μmol/L) inhibited ICaL by 33.4%. In the presence of ATX (3 nmol/L), the rate and amplitude of cell shortening and relaxation, the diastolic [Ca(2+)]i, and the rate and amplitude of [Ca(2+)]i rise and decay were significantly increased, which were reverted to control levels by tetrodotoxin (TTX, 2 μmol/L) or by ketamine (40 μmol/L). Ketamine protects isolated rabbit ventricular myocytes against [Ca(2+)]i overload by inhibiting INaL and ICaL.

Crocin, a carotenoid component of Crocus cativus, exerts inhibitory effects on L-type Ca(2+) current, Ca(2+) transient, and contractility in rat ventricular myocytes.

Crocin, a carotenoid component of Crocus sativus L. belonging to the Iridaceae family, has demonstrated cardioprotective effects. To investigate the cellular mechanisms of these cardioprotective effects, here we studied the influence of crocin on L-type Ca(2+)current (I(Ca-L)), intracellular Ca(2+) ([Ca(2+)]i), and contraction of isolated rat cardiomyocytes by using the whole-cell patch-clamp technique and video-based edge detection and dual excitation fluorescence photomultiplier systems. Crocin inhibited I(Ca-L) in a concentration-dependent manner with the half-maximal inhibitory concentration (IC50) of 45 μmol/L and the maximal inhibitory effect of 72.195% ± 1.54%. Neither current-voltage relationship of I(Ca-L), reversal potential of I(Ca-L), nor the activation/inactivation of I(Ca-L) was significantly changed. Crocin at 1 μmol/L reduced cell shortening by 44.64% ± 2.12% and the peak value of the Ca(2+) transient by 23.66% ± 4.52%. Crocin significantly reduced amplitudes of myocyte shortening and [Ca(2+)]i with an increase in the time to reach 10% of the peak (Tp) and a decrease in the time to 10% of the baseline (Tr). Thus, the cardioprotective effects of crocin may be attributed to the attenuation of [Ca(2+)]i through the inhibition of I(Ca-L) in rat cardiomyocytes and negative inotropic effects on myocardial contractility.

Abstract 156: Hydrogel Mattress, an in vitro Platform to Enhance Maturation and Evaluate Contractile Function of Individual hiPSC-CMs

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) have great potential as tools for human heart disease modeling and drug discovery. However, their contractile properties have not been routinely evaluated; as current methods are not accessible for most laboratories. We sought to develop a more efficient method to evaluate hiPSC-CM mechanical properties, at the single cell level. Individual hiPSC-CMs were cultured on a hydrogel based platform, termed the “hydrogel mattress,” and their cellular contractile properties evaluated using video-based edge detection. We found that hiPSC-CMs maintained on the mattress reproducibly exhibited robust cell shortening, in dramatic contrast to hiPSC-CMs maintained in a standard manner. We further found that contraction and peak cell shortening amplitude of hiPSC-CMs on mattress was comparable to that of freshly isolated adult ventricular mouse CM. Importantly, hiPSC-CMs maintained on the mattress exhibited several characteristics of a native CM, in terms of myocyte elongation, calcium handling and pharmacological response. Finally, using this platform, we could calculate the traction force generated by individual CMs. In summary, the Hydrogel mattress platform is a simple and reliable in vitro platform that not only enables the quantification of contractile performance of isolated hiPSC-CMs, but also enhances CM maturation. This flexible platform can be extended to in vitro disease modeling, drug discovery and cardiotoxicity testing.

MicroRNA-223 Displays a Protective Role Against Cardiomyocyte Hypertrophy by Targeting Cardiac Troponin I-Interacting Kinase

Background/Aims: MicroRNAs play regulatory role in cardiovascular disease. MicroRNA-223 (miR-223) was found to be expressed abundantly in myocardium. TNNI3K, a novel cardiac troponin I (cTnI)-interacting and cardiac hypertrophy related kinase, is computationally predicted as a potential target of miR-223. This study was designed to investigate the cellular and molecular effects of miR-223 on cardiomyoctye hypertrophy, focusing on the role of TNNI3K. Methods: Neonatal rat cardiomyocytes (CMs) were cultured, and CMs hypertrophy was induced by endothelin-1 (ET-1). In vivo cardiac hypertrophy was induced by transverse aorta constriction (TAC) in rats. Expression of miR-223 in CMs and myocardium was detected by real-time PCR (RT-PCR). MiR-223 and TNNI3K were overexpressed in CMs via chemically modifed sense RNA (miR-223 mimic) transfection or recombinant adenovirus infection, respectively. Cell size was measured by surface area calculation using fluorescence microscopy after anti-α-actinin staining. Expression of hypertrophy-related genes was detected by RT-PCR. The protein expression of TNNI3K and cTnI was determined by Western blots. Luciferase assay was employed to confirm the direct binding of miR-223 to the 3'UTR of TNNI3K mRNA. Intracellular calcium was measured by sensitive fluorescent indicator (Furo-2). Video-based edge detection system was employed to measure cardiomyocyte contractility. Results: MiR-223 was downregulated in ET-1 induced hypertrophic CMs and in hypertrophic myocardium compared with respective controls. MiR-223 overexpression in CMs alleviated ET-1 induced hypertrophy, evidenced by smaller cell surface area and downregulated ANP, α-actinin, Myh6 and Myh7 expression. Luciferase reporter gene assay showed that TNNI3K serves as a direct target gene of miR-223. In miR-223-overexpressed CMs, the protein expression of TNNI3K was significantly downregulated. MiR-223 overexpression also rescued the upregulated TNNI3K expression in hypertrophic CMs. Furthermore, cTnI phosphorylation was downregulated post miR-223 overexpression. Ad.rTNNI3K increased intracellular Ca²⁺ concentrations and cell shortening in CMs, while miR-223 overexpression significantly rescued these hypertrophic effects. Conclusion: By direct targeting TNNI3K, miR-223 could suppress CMs hypertrophy via downregulating cTnI phosphorylation, reducing intracellular Ca²⁺ and contractility of CMs. miR-223 / TNNI3K axis may thus be major players of CMs hypertrophy.

Salvia miltiorrhiza (Danshen) inhibits L-type calcium current and attenuates calcium transient and contractility in rat ventricular myocytes

Ethnopharmacological relevanceSalvia miltiorrhiza (SM, Danshen), a traditional Chinese herbal drug, has been widely used for hundreds of years to treat coronary artery disease. Materials and methodsWe studied the effects of SM on the L-type Ca2+ current (ICa-L) with whole-cell patch-clamp technique in rat ventricular myocytes, and its influence on Ca2+ transient and contractility using video-based edge detection and dual excitation fluorescence photomultiplier systems as well. ResultsExposure to SM solution caused a concentration- and voltage-dependent blockade of ICa-L, and the dose of SM solution (10g/l) decreased the maximal inhibitory effect of 35.2±1.2%. However, SM solution did not significantly change the current–voltage relationship or reversal potential of ICa-L, nor did it altered the activation and inactivation gating properties of cardiac Ca2+ channels. Meanwhile, SM decreased the amplitude of myocyte shortening and the peak value of Ca2+ transient with a significant decrease in the time to 90% of the baseline (Tr), but the time to 10% of the peak (Tp) was not dramatically prolonged. ConclusionsThe results indicated that SM significantly inhibited L-type Ca2+ channels, decreased [Ca2+]i and contractility in adult rat cardiac myocytes. These findings may be relevant to the cardioprotective efficacy of SM.

Inhibitory effect of cinobufagin on L-type Ca2+ currents, contractility, and Ca2+ homeostasis of isolated adult rat ventricular myocytes.

Cinobufagin (CBG), a major bioactive ingredient of the bufanolide steroid compounds of Chan Su, has been widely used to treat coronary heart disease. At present, the effect of CBG on the L-type Ca2+ current (I Ca-L) of ventricular myocytes remains undefined. The aim of the present study was to characterize the effect of CBG on intracellular Ca2+ ([Ca2+]i) handling and cell contractility in rat ventricular myocytes. CBG was investigated by determining its influence on I Ca-L, Ca2+ transient, and contractility in rat ventricular myocytes using the whole-cell patch-clamp technique and video-based edge-detection and dual-excitation fluorescence photomultiplier systems. The dose of CBG (10−8 M) decreased the maximal inhibition of CBG by 47.93%. CBG reduced I Ca-L in a concentration-dependent manner with an IC50 of 4 × 10−10 M, upshifted the current-voltage curve of I Ca-L, and shifted the activation and inactivation curves of I Ca-L leftward. Moreover, CBG diminished the amplitude of the cell shortening and Ca2+ transients with a decrease in the time to peak (Tp) and the time to 50% of the baseline (Tr). CBG inhibited L-type Ca2+ channels, and reduced [Ca2+]i and contractility in adult rat ventricular myocytes. These findings contribute to the understanding of the cardioprotective efficacy of CBG.

The effect of ligustrazine on L-type calcium current, calcium transient and contractility in rabbit ventricular myocytes

Ethnopharmacological relevanceLigustrazine, the biologically active ingredient isolated from a popular Chinese medicinal plant, Ligusticum chuanxiong Hort. (Umbelliferae), has been used effectively to treat ischemic heart diseases, cerebrovascular and thrombotic vascular diseases since the 1970s. Materials and methodsAt present, the effect of ligustrazine on L-type calcium current (ICa-L) of ventricular myocytes remains controversial. In this study, we use the whole-cell patch-clamp techniques and video-based edge detection and dual excitation fluorescence photomultiplier systems to study the effects of ligustrazine on ICa-L, and calcium transient and contractility in rabbit ventricular myocytes in the absence and presence of isoprenaline (ISO). ResultsLigustrazine (5μM) in low concentration did not affect ICa-L (P>0.05), higher concentrations of this drug (10, 20, 40, 80μM) inhibited ICa-L in a concentration-dependent manner and reduced ICa-L by 9.6±2.9%, 21.0±4.3%, 33.9±4.3%, and 51.6±7.3%, respectively. Under normal conditions, ligustrazine (40μΜ) reduced baseline of fura-2 fluorescence intensities (FFI, 340/380 ratio), namely diastolic calcium concentration, changes in FFI (ΔFFI, 340/380 ratio) and maximal velocity of Ca2+ rise and decay (340/380ratio/ms) by 6.3%, 26.1%, 25.2%, and 26.5%, and decreased sarcomere peak shorting (PS) and maximal velocity of shorting and relengthening by 36.4%, 31.9%, and 25.0%, respectively. Similarly, ligustrazine (40μM) reduced baseline FFI, ΔFFI, and maximal velocity of Ca2+ rise and decay by 14.1%, 51.1%, 35.2%, and 41.1%, and reduced sarcomere PS and maximal velocity of shorting and relengthening by 38.6%, 50.0% and 39.1%, respectively, in the presence of ISO. ConclusionsLigustrazine not only significantly inhibits ICa-L in a concentration-dependent manner but also suppressed calcium transient and contraction in the absence and presence of ISO.

Hyperkalemic Cardioplegia-Induced Myocyte Swelling and Contractile Dysfunction: Prevention by Diazoxide

Hyperkalemic cardioplegia (9 degrees C) results in significant myocyte swelling and reduced contractility, representing a possible mechanism of myocardial stunning. Adenosine triphosphate-sensitive potassium channel (KATP) openers have been shown to ameliorate stunning. This study evaluated the hypothesis that a KATP opener would prevent hyperkalemic cardioplegia-induced myocyte swelling and reduced contractility. Isolated rabbit myocytes were perfused with 37 degrees C Tyrode's solution for 20 minutes, followed by test solution (9 degrees C or 37 degrees C) including control Tyrode's, Tyrode's + 100 micromol/L diazoxide (KATP opener), St. Thomas's solution; or 9 degrees C St. Thomas's + 100 micromol/L diazoxide or St. Thomas's + 100 micromol/L diazoxide + 20 micromol/L HMR1098 or 50 micromol/L 5-hydroxydeconoate (KATP blockers) for 20 minutes (n = 8 per group). Myocytes were then reexposed to 37 degrees C Tyrode's solution for 20 minutes. Volume and contractility were measured by videomicroscopy and video-based edge detection, respectively. St. Thomas's solution (9 degrees C) caused significant myocyte swelling and associated reduced contractility (p < 0.05). The addition of diazoxide abolished myocyte swelling (p < 0.0001), and eliminated the associated reduced contractility (p < 0.05). Findings were unchanged by the addition of HMR 1098 and 5-hydroxydeconoate. Diazoxide prevented myocyte swelling and reduced contractility secondary to hyperkalemic cardioplegia, and this was unchanged by the addition of either KATP channel blocker. Prevention of myocyte swelling was associated with improved contractility, consistent with the hypothesis that myocyte swelling may be a mechanism of myocardial stunning. Diazoxide may play a role in myocyte volume homeostasis by means of a mechanism separate from opening the KATP channel.

Adenovirus gene transfer of recombinant endothelial nitric oxide synthase enhances contractile function in ventricular myocytes.

eNOS is expressed in cardiac myocytes and plays an important role in cardiac contractile function. This study was designed to determine whether ex vivo eNOS gene transfer in ventricular myocytes affects cardiac contractile function. Replication-incompetent adenoviral vectors encoding eNOS or marker gene beta-galactosidase (LacZ) were transduced into adult rat ventricular myocytes at an MOI of 10, 50, or 100 for 36 hours. Mechanical and intracellular Ca2+ properties of myocytes were evaluated by video-based edge detection and fura-2 fluorescence. NOS protein expression and activity were assessed by Western blot and 3H-arginine to 3H-citrulline assay. Myocytes transduced with eNOS but not LacZ displayed enhanced eNOS but not iNOS expression associated with elevated NOS activity. Myocytes transduced with eNOS exhibited significantly elevated peak shortening and velocity of shortening/relengthening associated with enhanced basal as well as electrically stimulated rise of intracellular Ca2+ compared with control or LacZ groups. The durations of shortening and relengthening were comparable in all groups. The eNOS-induced mechanical effects were paralleled with elevated phosphorylation of Akt. Furthermore, the phosphatidylinositol-3 (PI-3) kinase inhibitors wortmannin and LY294002 prevented eNOS-induced mechanical effects. These results revealed that gene transfer of eNOS directly promotes cardiomyocyte contractile function and intracellular Ca2+ handling, suggesting therapeutic potential of eNOS gene transfer.

Leptin attenuates cardiac contraction in rat ventricular myocytes. Role of NO.

Obesity is commonly associated with impaired myocardial contractile function. However, a direct link between these 2 states has not yet been established. There has been an indication that leptin, the product of the human obesity gene, may play a role in obesity-related metabolic and cardiovascular dysfunctions. The purpose of this study was to determine whether leptin exerts any direct cardiac contractile action that may contribute to altered myocardial function. Ventricular myocytes were isolated from adult male Sprague-Dawley rats. Contractile responses were evaluated by use of video-based edge detection. Contractile properties analyzed in cells electrically stimulated at 0.5 Hz included peak shortening, time to 90% peak shortening, time to 90% relengthening, and fluorescence intensity change. Leptin exhibited a dose-dependent inhibition in myocyte shortening and intracellular Ca(2+) change, with maximal inhibitions of 22.4% and 26.2%, respectively. Pretreatment with the NO synthase inhibitor N:(omega)-nitro-L-arginine methyl ester (L-NAME, 100 micromol/L) blocked leptin-induced inhibition of both peak shortening and fluorescence intensity change. Leptin also stimulated NO synthase activity in a time- and concentration-dependent manner, as reflected in the dose-related increase in NO accumulation in these cells. Addition of an NO donor (S-nitroso-N-acetyl-penicillamine [SNAP]) to the medium mimicked the effects of leptin administration. In summary, this study demonstrated a direct action of leptin on cardiomyocyte contraction, possibly through an increased NO production. These data suggest that leptin may play a role in obesity-related cardiac contractile dysfunction.