Neonatal Myocytes Research Articles

β-Adrenergic Signaling Inhibits G q -Dependent Protein Kinase D Activation by Preventing Protein Kinase D Translocation

Rationale: Both β-adrenergic receptor (β-AR) and G q -coupled receptor (G q R) agonist–driven signaling play key roles in the events, leading up to and during cardiac dysfunction. How these stimuli interact at the level of protein kinase D (PKD), a nodal point in cardiac hypertrophic signaling, remains unclear. Objective: To assess the spatiotemporal dynamics of PKD activation in response to β-AR signaling alone and on coactivation with G q R-agonists. This will test our hypothesis that compartmentalized PKD signaling reconciles disparate findings of PKA facilitation and inhibition of PKD activation. Methods and Results: We report on the spatial and temporal profiles of PKD activation using green fluorescent protein-tagged PKD (wildtype or mutant S427E) and targeted fluorescence resonance energy transfer–based biosensors (D-kinase activity reporters) in adult cardiomyocytes. We find that β-AR/PKA signaling drives local nuclear activation of PKD, without preceding sarcolemmal translocation. We also discover pronounced interference of β-AR/cAMP/PKA signaling on G q R-induced translocation and activation of PKD throughout the cardiomyocyte. We attribute these effects to direct, PKA-dependent phosphorylation of PKD-S427. We also show that phosphomimetic substitution of S427 likewise impedes G q R-induced PKD translocation and activation. In neonatal myocytes, S427E inhibits G q R-evoked cell growth and expression of hypertrophic markers. Finally, we show altered S427 phosphorylation in transverse aortic constriction–induced hypertrophy. Conclusions: β-AR signaling triggers local nuclear signaling and inhibits G q R-mediated PKD activation by preventing its intracellular translocation. PKA-dependent phosphorylation of PKD-S427 fine-tunes the PKD responsiveness to G q R-agonists, serving as a key integration point for β-adrenergic and G q -coupled stimuli.

Selective hydrophilic modification of Parylene C films: a new approach to cell micro-patterning for synthetic biology applications

We demonstrate a simple, accurate and versatile method to manipulate Parylene C, a material widely known for its high biocompatibility, and transform it to a substrate that can effectively control the cellular microenvironment and consequently affect the morphology and function of the cells in vitro. The Parylene C scaffolds are fabricated by selectively increasing the material's surface water affinity through lithography and oxygen plasma treatment, providing free bonds for attachment of hydrophilic biomolecules. The micro-engineered constructs were tested as culture scaffolds for rat ventricular fibroblasts and neonatal myocytes (NRVM), toward modeling the unique anisotropic architecture of native cardiac tissue. The scaffolds induced the patterning of extracellular matrix compounds and therefore of the cells, which demonstrated substantial alignment compared to typical unstructured cultures. Ca2+ cycling properties of the NRVM measured at rates of stimulation 0.5–2 Hz were significantly modified with a shorter time to peak and time to 90% decay, and a larger fluorescence amplitude (p < 0.001). The proposed technique is compatible with standard cell culturing protocols and exhibits long-term pattern durability. Moreover, it allows the integration of monitoring modalities into the micro-engineered substrates for a comprehensive interrogation of physiological parameters.

Sarcomere length nanometry in rat neonatal cardiomyocytes expressed with α-actinin–AcGFP in Z discs

Nanometry is widely used in biological sciences to analyze the movement of molecules or molecular assemblies in cells and in vivo. In cardiac muscle, a change in sarcomere length (SL) by a mere ∼100 nm causes a substantial change in contractility, indicating the need for the simultaneous measurement of SL and intracellular Ca(2+) concentration ([Ca(2+)]i) in cardiomyocytes at high spatial and temporal resolution. To accurately analyze the motion of individual sarcomeres with nanometer precision during excitation-contraction coupling, we applied nanometry techniques to primary-cultured rat neonatal cardiomyocytes. First, we developed an experimental system for simultaneous nanoscale analysis of single sarcomere dynamics and [Ca(2+)]i changes via the expression of AcGFP in Z discs. We found that the averaging of the lengths of sarcomeres along the myocyte, a method generally used in today's myocardial research, caused marked underestimation of sarcomere lengthening speed because of the superpositioning of different timings for lengthening between sequentially connected sarcomeres. Then, we found that after treatment with ionomycin, neonatal myocytes exhibited spontaneous sarcomeric oscillations (cell-SPOCs) at partial activation with blockage of sarcoplasmic reticulum functions, and the waveform properties were indistinguishable from those obtained in electric field stimulation. The myosin activator omecamtiv mecarbil markedly enhanced Z-disc displacement during cell-SPOC. Finally, we interpreted the present experimental findings in the framework of our mathematical model of SPOCs. The present experimental system has a broad range of application possibilities for unveiling single sarcomere dynamics during excitation-contraction coupling in cardiomyocytes under various settings.

Cx43 phosphorylation on S279/282 and intercellular communication are regulated by IP 3 /IP 3 receptor signaling

Inositol 1,4,5-trisphosphate receptor (IP3R) plays a pivotal role in the Ca2+ release process in a variety of cell types. Additionally, IP3R is distributed in ventricular intercalated discs, but its function(s) in this particular site remains unknown. Connexin (Cx43), the predominant gap junction (GJ) protein in ventricular myocardium, is linked to several signaling pathways that regulate Cx43 properties by (de)phosphorylation on multiple residues. Here, we investigated the regulatory role of IP3R in cell-cell communication and the mechanism(s) underlying this effect. In neonatal rat and adult mouse ventricular myocytes IP3R co-localized and co-immunoprecipitated with Cx43 in GJ plaques detected by immunostaining and western blot assays. Blocking IP3R with antagonists or silencing pan-IP3R expression with shRNA hindered the 6-carboxyfluorescein (6-CFDA) diffusion through GJs and desynchronized Ca2+ transients among confluent neonatal myocytes in culture, whereas stimulation of IP3R with IP3 ester or ATP exerted the opposite effect. Likewise, 6-CFDA propagation through GJs was modulated by IP3R activation or inhibition in cell pairs of isolated adult cardiomyocytes. Furthermore, IP3R activation or IP3R suppression promoted or suppressed, respectively, Cx43 phosphorylation on S279/282. Site-directed mutagenesis indicated that expression of a mutant Cx43-S282A (alanine) inhibited S279/282 phosphorylation and GJ permeability, while the S279A mutant showed the opposite effect in ventricular myocytes. Expression of these mutants in HEK293 cells revealed that cells with a dual S279/282 mutation failed to express exogenous Cx43, whereas cells with a single S279 or S282 mutation displayed Cx43 overexpression with increased phosphorylation of S279/282 and promotion of intercellular communication. These results demonstrated, for the first time, that IP3R physically interacts with Cx43 and participates in the regulation of Cx43 phosphorylation on S279/282, thereby affecting GJ intercellular communication in ventricular myocytes.

Sarcomere Length Nanometry in Cardiomyocytes Expressed with α-Actinin-AcGFP in Z-Discs

Nanometry is widely used in today's biological sciences to analyze the movement of molecules or molecular assemblies in cells and in vivo. In cardiac muscle, a change in sarcomere length (SL) by a mere ∼100 nm causes a dramatic change in contractility (i.e., the Frank-Starling mechanism), indicating the need for the simultaneous measurement of SL and intracellular Ca2+ concentration ([Ca2+]i) in cardiomyocytes at high spatial and temporal resolution. To accurately analyze the motion of individual sarcomeres with nanometer precision during excitation-contraction coupling, we in the present study applied nanometry techniques to primary-cultured rat neonatal cardiomyocytes. First, we developed an experimental system for simultaneous nano-scale analysis of single sarcomere dynamics and [Ca2+]i changes via the expression of AcGFP in Z-discs. We found that the averaging of the lengths of sarcomeres along the myocyte caused marked underestimation of sarcomere lengthening speed due to superpositioning of different timings for lengthening between sequentially connected sarcomeres. Then, we found that following the treatment with ionomycin, neonatal myocytes exhibited spontaneous sarcomeric oscillations (Cell-SPOC) at partial activation with blockage of sarcoplasmic reticulum functions, and the waveform properties were indistinguishable from those obtained in electric field stimulation. The myosin activator, omecamtiv mecarbil, markedly enhanced Z-disc displacement during Cell-SPOC. Finally, we interpreted the present experimental findings in the framework of our mathematical model of spontaneous sarcomeric oscillations (Sato, K. et al., Prog Biophys Mol Biol. 2011; Sato, K. et al., Phys. Rev. Lett. 2013). The present experimental system has a broad range of application possibilities for unveiling single sarcomere dynamics during excitation-contraction coupling in cardiomyocytes under various settings.

Functional Interaction with Filamin a Enhances Atrial-Specific Small Conductance Ca2 Activated K+ Channel (SK2) Surface Membrane Expression

For ion channels to function properly, a precise number of channel proteins need to be trafficked to exact locations on the cell surface membrane. Small-conductance, Ca2+-activated K+ channels (SK) are predominantly expressed in the atria and their role has been implicated in atrial fibrillation (AF).Using yeast two-hybrid screen against human heart library, we identify filamin A (FLNA) as a putative interacting protein with SK2 channel. Patch-clamp recordings and immunofuorescence studies in neonatal and adult cardiac myocytes as well as HEK293 cells suggest the interaction leads to an increase in SK2 membrane localization. SiRNA knockdown of FLNA in neonatal myocytes results in a decrease in the membrane localization of SK2 channels. Additionally, the calcium dependency of SK2 channel membrane expression was examined using Total Internal Reflection Microscopy (TIRF-M) and immunofluorescence microscopy. Importantly, intracellular Ca2+ (Ca2+i) plays a critical role in the membrane localization of SK2 channel when the channel is co-expressed with α-actinin2, another cytoskeletal protein which we have previously shown to interact with SK2 channel. In conclusion, FLNA is a regulator of SK2 channel expression. Moreover, SK2 membrane expression is critically dependent on Ca2+i. An increase in Ca2+i, for example, during AF, is predicted to result in an increase in SK2 channel expression leading to shortening of the action potentials.

Mechanisms With Clinical Implications for Atrial Fibrillation–Associated Remodeling: Cathepsin K Expression, Regulation, and Therapeutic Target and Biomarker

BackgroundThe cysteine protease cathepsin K (CatK) has been implicated in the pathogenesis of cardiovascular disease. We sought to determine the link between atrial fibrillation (AF) and plasma CatK levels and to investigate the expression of and therapeutic target for CatK in vivo and in vitro.Methods and ResultsPlasma CatK and extracellular matrix protein peptides (intact procollagen type I of N‐terminal propeptide; carboxyl‐terminal telopeptide of type I collagen [ICTP]) were measured in 209 consecutive patients with AF (paroxysmal AF, 146; persistent AF, 63) and 112 control subjects. In addition, the regulation of CatK expression was investigated in vivo and vitro. Patients with AF had higher plasma CatK and ICTP levels than did control subjects. Patients with persistent AF had higher levels of plasma CatK and ICTP than did patients with paroxysmal AF. CatK was correlated with ICTP concentration and left atrial diameter in all subjects. In rabbits, superoxide production, CatK activity, fibrosis, and the levels of atrial tissue angiotensin II, angiotensin type 1 receptor, gp91phox, phospho‐p38 mitogen‐activated protein kinase, and CatK were greater in those with tachypacing‐induced AF than in controls, and these changes were reversed with angiotensin type 1 receptor antagonist. Olmesartan and mitogen‐activated protein kinase inhibitor decreased the CatK expression induced by angiotensin II in rat neonatal myocytes.ConclusionsThese data indicated that increased plasma CatK levels are linked with the presence of AF. Angiotensin type 1 receptor antagonist appears to be effective in alleviating atrial fibrosis in a rabbit AF model, partly reducing angiotensin type 1 receptor‐p38mitogen‐activated protein kinase‐dependent and ‐independent CatK activation, thus preventing AF.

PRAS40 prevents development of diabetic cardiomyopathy and improves hepatic insulin sensitivity in obesity

Diabetes is a multi-organ disease and diabetic cardiomyopathy can result in heart failure, which is a leading cause of morbidity and mortality in diabetic patients. In the liver, insulin resistance contributes to hyperglycaemia and hyperlipidaemia, which further worsens the metabolic profile. Defects in mTOR signalling are believed to contribute to metabolic dysfunctions in diabetic liver and hearts, but evidence is missing that mTOR activation is causal to the development of diabetic cardiomyopathy. This study shows that specific mTORC1 inhibition by PRAS40 prevents the development of diabetic cardiomyopathy. This phenotype was associated with improved metabolic function, blunted hypertrophic growth and preserved cardiac function. In addition PRAS40 treatment improves hepatic insulin sensitivity and reduces systemic hyperglycaemia in obese mice. Thus, unlike rapamycin, mTORC1 inhibition with PRAS40 improves metabolic profile in diabetic mice. These findings may open novel avenues for therapeutic strategies using PRAS40 directed against diabetic-related diseases.

Abstract 261: Human IPS-Derived Cardiac Myocytes (hiPSC-CMs): How Good Are They as a Model for Cardiac Pacing and E-C Coupling?

Derivation of cardiomyocytes from human induced pluripotent stem cells has opened a new field of biology where hiPSC-CM are being used as electrophysiological models of human cardiovascular physiology and pathology. Whether hiPS-CM represent also reliable Ca 2+ signaling and pacemaking models for the mammalian myocytes remains to be determined. Here we evaluate the EC-coupling and spontaneous pacemaking properties of human iPS-CM in detail and compare them to those of native mammalian myocytes. iPS-CMs, dissociated after ~40 days of differentiation and voltage-clamped at -50 mV, showed spontaneous beating and Ca 2+ oscillations that activated in-phase I NCX oscillations at holding potentials between -60 and +20mV, while I f activated negative to -75mV. Withdrawal of [Ca 2+ ] o and application of NCX-blocker (KBR- 7943) or tetracaine rapidly and reversibly inhibited spontaneous Ca 2+ -oscillations. Nifedipine and NO-synthase inhibitor L-NAME failed to alter spontaneous beating. Identical sets of data as these were also obtained from neonatal rat myocytes (NRM), suggesting that SR Ca 2+ release and uptake, and not I f , were responsible for spontaneous beating in NRM and hiPS-CM. Ca 2+ signaling parameters of hiPS-CM were also similar to those of adult atrial myocytes with Ca 2+ currents averaging ~8 pA/pF and I Ca -induced Ca 2+ -transients having a bell-shaped voltage-dependence similar to that of I Ca , consistent with Ca 2+ -induced Ca 2+ -release (CICR) mechanism. The ratio of I Ca -activated to caffeine-triggered Ca 2+ -transients was ~0.3, similar to the value in rat atria, but significantly smaller than the value of &gt;0.8 in ventricle. The gain of CICR was voltage-dependent as in adult cardiomyocytes. Adrenergic agonists enhanced I Ca , but elevated diastolic Ca 2+ . Ca 2+ -sparks were sporadic and brief in duration (&lt; 25ms). Our data suggest that hiPS-CM have all the specific characteristics of adult cardiomyocytes and the mechanisms of their spontaneous pacing are similar to those found in NRM, and involve Ca 2+ cross-talk between NCX, RyR/SR, and possibly mitochondria.

Abstract 081: Liganded Cannabinoid Receptors Suppress Cardiac Myocyte Hypertrophy by AMP-activated protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS) signaling

Endocannabinoids are bioactive lipids that signal through CB1 and CB2 cannabinoid receptors. Despite an effort to avoid the psychoactive side effects mediated by central CB1 receptors, we previously found that selective activation of CB2 receptors is not sufficient to prevent cardiac myocyte hypertrophy. Thus, the objective of this study is to determine the effects of CB13, a peripherally-restricted agonist of CB1/CB2 receptors, on hypertrophy. The effects of CB-13 on endothelin-1 (ET1)-induced hypertrophy were determined using isolated neonatal rat myocytes. Hypertrophic indicators included myocyte enlargement, protein synthesis by [3H]-leucine incorporation, and fetal gene expression as brain natriuretic peptide (BNP) mRNA levels. AMPK and eNOS signaling were assessed by immunoblotting. ET1 increased myocyte size (122±4% vs. control; p&lt;0.01), BNP expression (426±88% vs. control; p&lt;0.01) and [3H]-leucine incorporation (147±13% vs. control; p&lt;0.05). CB13 attenuated all three hypertrophic indicators (respectively, 104±3%, 210±32%, 90±8% vs. control; not significant). We next queried whether the anti-hypertrophic actions of CB13 were mediated by AMPK. CB13 increased total expression (222±45% vs. control; p&lt;0.05) and phosphorylation (354±58% vs. control; p&lt;0.01) of AMPK. Also, pretreatment with a chemical inhibitor of AMPK, compound C, attenuated the anti-hypertrophic actions of CB-13. AMPK is a regulator of cellular energy, so we determined that ET-1-induced mitochondrial depolarization, as assessed using the potential-sensitive dye, JC-1, was prevented by CB-13. In addition, CB-13 increased eNOS phosphorylation (237±51%; p&lt;0.01), which suggests that AMPK-eNOS crosstalk, through anti-growth nitric oxide signaling, plays a role. These findings support the notion that agonism of CB1 and CB2 receptors by CB13, a synthetic endocannabinoid with poor brain-blood barrier penetration, prevents hypertrophy and mitochondrial dysfunction. These CB13 actions rely on AMPK signaling, and are associated with eNOS activation. In conclusion, cannabinoid-based treatment of heart disease remains a viable goal with therapeutic potential and warrants further study.

Abstract 355: In Vivo Targeting Of GTF2B Selectively Inhibits Transcription Of Cardiomyopathy-related Genes And Partially Blunts Development Of Cardiac Hypertrophy

Transcriptional profiling of cardiac genome during hypertrophy identified two categories of genes with distinct modes of regulation. The first set of genes involved in the cells essential functions (e.g. RNA splicing) and whose transcription is expected to be incremental and contribute to the increasing cardiac mass is regulated by promoter clearance of RNA polymerase II (pol II). On the other hand, the second set that include genes with specialized function and show a robust increase in expression upon growth stimulus (cytoskeletal, extracellular matrix) are regulated by de novo pol II recruitment to promoters. Our goal was to identify the transcriptional mechanisms that distinguish these two sets of genes and then to selectively inhibit those that participate in contractile dysfunction, while preserving the expression of genes necessary for essential functions. General Transcription factor IIB (GTF2B), is one of the essential components of transcription machinery and is required for pol II recruitment. Thus, we hypothesized that inhibition of GTF2B would result in inhibition of only the specialized genes, sparing the essential genes. Our in vitro results with shRNA mediated inhibition of GTF2B in hypertrophying neonatal myocytes showed decreased expression of genes that required de novo pol II recruitment for transcription (eg. ACTA1), while no change was observed in the genes regulated by promoter clearance of pol II (Vdac1). Similarly, preliminary results with in vivo knockdown of GTF2B (~80% reduction in mRNA and ~36% in protein) via intravenous injection of modified antisense oligo in mice subjected to transaortic coarctation (TAC) showed inhibition of only cardiomyopathy-related genes that require pol II recruitment (ANF), while expression of essential genes (Vdac1) remained unchanged. Inhibition of GTF2B restricted increase in TAC-induced heart wt to 9%, compared to 29% in TAC hearts with control oligo. Echocardiography showed partial normalization of ejection fraction with GTF2B inhibitor during TAC from 61.5% to 66.4% compared to sham hearts with 71%. Thus, we conclude that by targeting GTF2B we can selectively restrict the expression of detrimental genes during hypertrophy, thereby delaying the onset of cardiac dysfunction and failure

T-type Ca2 + channels regulate the exit of cardiac myocytes from the cell cycle after birth

T-type Ca2+ channels (TTCCs) are expressed in the fetal heart and then disappear from ventricular myocytes after birth. The hypothesis examined in this study was the α1G TTCCs' influence in myocyte maturation and their rapid withdrawal from the cell cycle after birth. MethodsCardiac myocytes were isolated from neonatal and adult wild type (WT), α1G−/− and α1G over expressing (α1GDT) mice. Bromodeoxyuridine (BrdU) uptake, myocyte nucleation, cell cycle analysis, and T-type Ca2+ currents were measured. ResultsAll myocytes were mono-nucleated at birth and 35% of WT myocytes expressed functional TTCCs. Very few neonatal myocytes had functional TTCCs in α1G−/− hearts. By the end of the first week after birth no WT or α1G−/− had functional TTCCs. During the first week after birth about 25% of WT myocytes were BrdU+ and became bi-nucleated. Significantly fewer α1G−/− myocytes became bi-nucleated and fewer of these myocytes were BrdU+. Neonatal α1G−/− myocytes were also smaller than WT. Adult WT and α1G−/− hearts were similar in size, but α1G−/− myocytes were smaller and a greater % were mono-nucleated. α1G over expressing hearts were smaller than WT but their myocytes were larger. ConclusionsThe studies performed show that loss of functional TTCCs is associated with bi-nucleation and myocyte withdrawal from the cell cycle. Loss of α1G TTCCs slowed the transition from mono- to bi-nucleation and resulted in an adult heart with a greater number of small cardiac myocytes. These results suggest that TTCCs are involved in the regulation of myocyte size and the exit of myocytes from the cell cycle during the first week after birth.

Hyperpolarization‐Activated Cyclic Nucleotide‐Gated Channels and Ventricular Arrhythmias in Heart Failure: A Novel Target for Therapy?

Arrhythmias are a common and often fatal complication of heart failure accounting for 50% to 70% of cardiac deaths due to tachyarrhythmic mechanisms.[1][1] In the failing heart, disturbances in cardiac rhythm are the likely result of cardiac remodeling consisting of structural and/or

Meis1 regulates postnatal cardiomyocyte cell cycle arrest

The neonatal mammalian heart is capable of substantial regeneration following injury through cardiomyocyte proliferation. However, this regenerative capacity is lost by postnatal day 7 and the mechanisms of cardiomyocyte cell cycle arrest remain unclear. The homeodomain transcription factor Meis1 is required for normal cardiac development but its role in cardiomyocytes is unknown. Here we identify Meis1 as a critical regulator of the cardiomyocyte cell cycle. Meis1 deletion in mouse cardiomyocytes was sufficient for extension of the postnatal proliferative window of cardiomyocytes, and for re-activation of cardiomyocyte mitosis in the adult heart with no deleterious effect on cardiac function. In contrast, overexpression of Meis1 in cardiomyocytes decreased neonatal myocyte proliferation and inhibited neonatal heart regeneration. Finally, we show that Meis1 is required for transcriptional activation of the synergistic CDK inhibitors p15, p16 and p21. These results identify Meis1 as a critical transcriptional regulator of cardiomyocyte proliferation and a potential therapeutic target for heart regeneration.

The dual‐specificity phosphatase 8 (Dusp8) regulates cardiac hypertrophic response in vitro and in vivo

BackgroundPhosphorylation and dephosphorylation of mitogen‐activated protein kinases (MAPKs) play important roles in regulating cardiac hypertrophy, remodeling, and heart failure. Dual‐specificity phosphatases (Dusps) directly dephosphorylate and inactivate each of the MAPK terminal kinases (ERK, p38, JNK) within 30 to 60 minutes activation. In this study, we generated mice and mouse embryonic fibroblasts (MEFs) lacking DUSP8 gene and determined the role of Dusp8 in regulating c‐Jun N‐terminal kinases (JNK1/2) signaling in neonatal myocyte culture and mouse heart. We hypothesized that knockout of Dusp8 will lead to increased JNK phosphorylation and decreased cardiac hypertrophy.MethodsDUSP8 null mice were generated using traditional gene‐targeting approach to delete exon 5 and 6. Phenylephrine was injected at final concentration of 10 mg/kg using 2‐month‐old mice.Results: Adenovirus mediated Dusp8 overexpression in cultured cardiomyocytes resulted in decreased JNK1/2 phosphorylation but not that of p38 and ERK1/2, indicating Dusp8 is specific for JNK dephosphorylation. In contrast, injection of Dusp8 null mice with phenylephrine led to sustained JNK phosphorylation compared to that of WT mice, which was also true for the JNK signaling in Dusp8 null MEF cells. Dusp8 null mice had reduced left ventricular weight/body weight ratio at both baseline and angiotensin II/phenylephrine stimulation condition. At cellular level, we found that overexpression of Dusp8 led to increased nuclear accumulation of NFAT and expression of hypertrophy gene ANF.ConclusionsThe results of the present study indicate that knockout of Dusp8 is cardioprotective through increasing JNK phosphorylation to prevent NAFT signaling induced cardiomyopathy.

Developmental Changes in Potassium Channel Expression in the Canine Heart: Implications for Sudden Infant Death Caused by Arrhythmias

Background and Rationale: It is believed that at least 10% of Sudden Infant Death syndrome cases are due to cardiac arrhythmia, however, developmental changes in the electrophysiological characteristics are not well defined in the higher mammalian heart. The present study examines the contribution of the inwardly rectifying K+ current (IK1), the transient outward K+ current (Ito) and the delayed rectifier K+ currents (IKr and IKs) to repolarization in the canine neonate myocardium.Methods and Results: Single cells were obtained from 2-3 week old canine neonate hearts subjected to enzymatic dispersion. Ventricular cells were isolated from both the left and right ventricles. Compared with adult ventricular cells, the size of neonate ventricular cells were approximately 6 times smaller. Action potential recordings showed a lack of phase 1 repolarization in neonatal myocytes. Accordingly, voltage clamp studies showed no Ito in neonatal myocytes. The inwardly rectifying potassium current IK1 was similar between neonate and adult. Measurement of the delayed rectifier(s) showed the presence of IKr, but not IKs. Additionally, mRNA levels for KCNQ1, KChIP2 and Kv4.3 were more than 2-fold lower in the neonatal heart compared with the adult heart;however, KCNH2 expression was unchanged.Conclusion: Repolarization in 2-3 week old canine neonate ventricle is due mainly to the presence of IK1 and IKr. Neither IKs nor Ito were recorded at this stage of development. These findings point to a reduced repolarization reserve in ventricular myocytes isolated from canine neonatal hearts when compared to those of adults. Our data may help explain why infants are more vulnerable to developing QT prolongation and arrhythmias.

Influence of cyclovirobuxine D on intracellular [Ca2 +] regulation and the expression of the calcium cycling proteins in rat myocytes

Our previous studies have shown that cyclovirobuxine D (CVB-D) ameliorated the cardiac function in heart failure rats. Considering the relationship between cardiac function and [Ca2+]i, and the role of calcium cycling on regulating [Ca2+]i, the present study was designed to evaluate the influence of CVB-D on the calcium transient of myocytes from neonatal rats and adult heart failure (HF) rats. The expression of calcium cycling proteins, including L-type calcium channel (LTCC), ryanodine receptor 2 (RYR2), sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) and sodium–calcium exchanger (NCX), were investigated to explore the underlying mechanism. CVB-D increased the intensity of calcium transient, accelerated the process of calcium transient and attenuation in the neonatal and adult myocytes. Furthermore, CVB-D shortened Tpeak and Tattenuation in the adult myocytes and slowed down the heart rate of neonatal myocytes. Besides, CVB-D increased the expression of RYR2 and SERCA2a, decreased the expression of NCX, but showed no significant effect on LTCC. Thus, it was concluded that CVB-D increased the release and uptake of Ca2+ in systolic and diastolic period, respectively. CVB-D might not only facilitate the utilization of intracellular Ca2+, but also prevent the loss of Ca2+.

Cardiac progenitor cells engineered with Pim-1 (CPCeP) develop cardiac phenotypic electrophysiological properties as they are co-cultured with neonatal myocytes

Stem cell transplantation has been successfully used for amelioration of cardiomyopathic injury using adult cardiac progenitor cells (CPC). Engineering of mouse CPC with the human serine/threonine kinase Pim-1 (CPCeP) enhances regeneration and cell survival in vivo, but it is unknown if such apparent lineage commitment is associated with maturation of electrophysiological properties and excitation–contraction coupling. This study aims to determine electrophysiology and Ca2+-handling properties of CPCeP using neonatal rat cardiomyocyte (NRCM) co-culture to promote cardiomyocyte lineage commitment. Measurements of membrane capacitance, dye transfer, expression of connexin 43 (Cx43), and transmission of ionic currents (ICa, INa) from one cell to the next suggest that a subset of co-cultured CPCeP and NRCM becomes connected via gap junctions. Unlike NRCM, CPCeP had no significant INa, but expressed nifedipine-sensitive ICa that could be measured more consistently with Ba2+ as permeant ion using ramp-clamp protocols than with Ca2+ and step-depolarization protocols. The magnitude of ICa in CPCeP increased during culture (4–7days vs. 1–3days) and was larger in co-cultures with NRCM and with NRCM-conditioned medium, than in mono-cultured CPCeP. ICa was virtually absent in CPC without engineered expression of Pim-1. Caffeine and KCl-activated Ca2+-transients were significantly present in co-cultured CPCeP, but smaller than in NRCM. Conversely, ATP-induced (IP3-mediated) Ca2+ transients were larger in CPCeP than in NRCM. INCX and IATP were expressed in equivalent densities in CPCeP and NRCM. These in vitro studies suggest that CPCeP in co-culture with NRCM: a) develop ICa current and Ca2+ signaling consistent with cardiac lineage, b) form electrical connections via Cx43 gap junctions, and c) respond to paracrine signals from NRCM. These properties may be essential for durable and functional myocardial regeneration under in vivo conditions.

Abstract 168: Does Calcium Influx Through TTCC Induce Cardiomyocyte Proliferation?

T-type Ca²[[Unable to Display Character: + ]] channels (TTCC) are primarily expressed in fetal/neonatal cardiomyocytes and their functional role is not well defined. Here we explored the idea that Ca 2+ influx through TTCC regulates myocyte proliferation. The fact that Mibefradil (TTCC and L-type calcium channel (LTCC) antagonist) inhibits smooth muscles proliferation supports this idea. Our hypothesis is: Blocking TTCC will reduce neonatal cardiomyocyte proliferation. Wild type (WT) and α1G TTCC knockout (α1G-/-) neonatal mice ventricular myocytes (NMVMs) were used. Patch clamp was used to measure TTCC. Flow cytometry was used to determine cell cycle distribution. 1. On day 1 (after birth), TTCC was detected in 35% WT NMVMs (n=31), whereas only 4% α1G-/- NMVMs have TTCC (n=25). On day 7, no TTCC was detected in both WT (n=16) and α1G-/- (n=27). 2. In vivo: On day 1 there’s no significant difference in cell cycle distribution: [88.2%±2.7% in G1/G0, 8.1%±2.8% in G2/M, 3.2%±0.1% in S; n=12] in WT vs. [90.6%±1.6% in G1/G0, 5.7%±1.5% in G2/M, 3.7%±0.3% in S; n=20] in α1G-/-. On day 7 there’s a significant difference: [52.3%±1.6% in G1/G0, 47.7%±1.6% in G2/M; n=28] in WT vs. [68.2%±2.1% in G1/G0, 31.9%±2.1% in G2/M; n=20] in α1G-/-, p&lt;0.05. 3. In vitro: Mono and binucleated myocytes percentage was measured: On day 1 there’s no significant difference. On day 7 there were more binucleated cells in WT: 51%±4% mono and 49%±4% binucleated in WT (n=495) vs. 80%±3% mono and 20%±3% binucleated in α1G-/- (n=1107), p&lt;0.05. Cell surface area (CSA) (um²) in α1G-/- (n=164) was smaller than WT (n=109): 860.3±323.3 in WT vs. 716.7±274.9 in α1G-/- in mono (p&lt;0.005), 1333.9±534.0 in WT vs. 1016.6±315.4 in α1G-/- in binucleated (p&lt;0.001). In 2-month old mice there’s a difference in percentage of mono and binucleated myocytes: [7.3%±3.7% mono, 92.7%±3.6% binucleated; n=687] in WT vs. [30.8%±6.1% mono, 69.2%±6.1% binucleated; n=793] in α1G-/-, p&lt;0.05. CSA in α1G-/- was smaller than in WT: 2267.4±1119.7 in WT vs. 1784.6±683.9 in α1G-/- in mononulceated (p&lt;0.0001), 2419.2±712.4 in WT vs. 2048.3±671.1 in α1G-/- in binucleated (p&lt;0.0001). There is large amount of DNA synthesis in NMVMs after birth, by day 7 most of the WT NMVMs are arrested in G2 and become binucleated. Myocyte without α1G TTCCs did not exit from the cell cycle normally.

Unusual localization and translocation of TRPV4 protein in cultured ventricular myocytes of the neonatal rat

TRPV4 protein forms a Ca2+-permeable channel that is sensitive to osmotic and mechanical stimuli and responds to warm temperatures, and expresses widely in various kinds of tissues. As for cardiac myocytes, TRPV4 has been detected only at the mRNA level and there were few reports about subcel-lular localization of the protein. The purpose of the present study was to investigate the expression profile of TRPV4 protein in cultured neonatal rat ventricular myocytes. Using Western blots, immunofluorescence, confocal microscopy and immuno-electron microscopy, we have shown that TRPV4 protein was predominantly located in the nucleus of cultured neonatal myocytes. Furthermore, cardiac myocytes responded to hypotonic stimulation by translocating TRPV4 protein out of the nucleus. The significance and mechanism concerning the unusual distribution and translocation of TRPV4 protein in cardiac myocytes remain to be clarified.