Adult Mouse Cardiomyocytes Research Articles

Fructose diet treatment in mice induces fundamental disturbance of cardiomyocyte Ca2+handling and myofilament responsiveness

High fructose intake has been linked to insulin resistance and cardiac pathology. Dietary fructose-induced myocardial signaling and morphological alterations have been described, but whether cardiomyocyte function is influenced by chronic high fructose intake is yet to be elucidated. The goal of this study was to evaluate the cardiomyocyte excitation-contraction coupling effects of high dietary fructose and determine the capacity for murine cardiomyocyte fructose transport. Male C57Bl/6J mice were fed a high fructose diet for 12 wk. Fructose- and control-fed mouse cardiomyocytes were isolated and loaded with the fura 2 Ca(2+) fluorescent dye for analysis of twitch and Ca(2+) transient characteristics (4 Hz stimulation, 37°C, 2 mM Ca(2+)). Myocardial Ca(2+)-handling protein expression was determined by Western blot. Gene expression of the fructose-specific transporter, GLUT5, in adult mouse cardiomyocytes was detected by real-time and conventional RT-PCR techniques. Diastolic Ca(2+) and Ca(2+) transient amplitude were decreased in isolated cardiomyocytes from fructose-fed mice relative to control (16 and 42%, respectively), coincident with an increase in the time constant of Ca(2+) transient decay (24%). Dietary fructose increased the myofilament response to Ca(2+) (as evidenced by a left shift in the shortening-Ca(2+) phase loop). Protein expression of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a), phosphorylated (P) phospholamban (Ser(16)), and P-phospholamban (Thr(17)) was reduced, and protein phosphatase 2A expression increased, in fructose-fed mouse hearts. Hypertension and cardiac hypertrophy were not evident. These findings demonstrate that fructose diet-associated myocardial insulin resistance induces profound disturbance of cardiomyocyte Ca(2+) handling and responsiveness in the absence of altered systemic loading conditions.

BIN1 is reduced and Cav1.2 trafficking is impaired in human failing cardiomyocytes

Heart failure is a growing epidemic, and a typical aspect of heart failure pathophysiology is altered calcium transients. Normal cardiac calcium transients are initiated by Cav1.2 channels at cardiac T tubules. Bridging integrator 1 (BIN1) is a membrane scaffolding protein that causes Cav1.2 to traffic to T tubules in healthy hearts. The mechanisms of Cav1.2 trafficking in heart failure are not known. To study BIN1 expression and its effect on Cav1.2 trafficking in failing hearts. Intact myocardium and freshly isolated cardiomyocytes from nonfailing and end-stage failing human hearts were used to study BIN1 expression and Cav1.2 localization. To confirm Cav1.2 surface expression dependence on BIN1, patch-clamp recordings were performed of Cav1.2 current in cell lines with and without trafficking-competent BIN1. Also, in adult mouse cardiomyocytes, surface Cav1.2 and calcium transients were studied after small hairpin RNA-mediated knockdown of BIN1. For a functional readout in intact heart, calcium transients and cardiac contractility were analyzed in a zebrafish model with morpholino-mediated knockdown of BIN1. BIN1 expression is significantly decreased in failing cardiomyocytes at both mRNA (30% down) and protein (36% down) levels. Peripheral Cav1.2 is reduced to 42% by imaging, and a biochemical T-tubule fraction of Cav1.2 is reduced to 68%. The total calcium current is reduced to 41% in a cell line expressing a nontrafficking BIN1 mutant. In mouse cardiomyocytes, BIN1 knockdown decreases surface Cav1.2 and impairs calcium transients. In zebrafish hearts, BIN1 knockdown causes a 75% reduction in calcium transients and severe ventricular contractile dysfunction. The data indicate that BIN1 is significantly reduced in human heart failure, and this reduction impairs Cav1.2 trafficking, calcium transients, and contractility.

Abstract 8393: IL-18 Binding Protein (IL-18BP) Inhibits β-AR Induced Cardiomyocyte Hypertrophy in vitro and Myocardial Hypertrophy i n vivo

Crosstalk between the sympathetic nervous system and proinflammatory cytokines such as interleukin-18 (IL-18) plays a key role in the pathophysiology of the hypertrophied failing heart. Importantly, IL-18 can directly promote cardiomyocyte hypertrophy. IL-18 binding protein (IL-18BP) is a natural inhibitor of IL-18, and counters its biological effects. While β-AR stimulation induces IL-18, it is not known whether it also regulates IL-18BP. Here we demonstrate that the β-AR agonist isoproterenol (ISO) upregulates IL-18BP mRNA and protein expression in adult mouse cardiomyocytes in β 2 -AR/Gi-dependent manner. We cloned mouse Il18bp 5’ cis -regulatory region, and identified several putative transcription factor-binding sites, including CREB, C/EBP, NF- k B, and Sp1. While forced expression of dnp65 or kdIKKβ, or pre-treatment with mithramycin (Sp1 inhibitor) had no significant effect, forced expression of dnCREB or C/EBPβ knockdown markedly attenuated ISO-induced Il18bp transcription. ISO induced Il18bp promoter-reporter activity, and the deletion or mutation of CREB and C/EBP motifs reduced the response. ISO induced CREB and C/EBPβ DNA-binding activity, reporter gene activation, and in vivo binding to Il18bp promoter. ISO induced CREB and C/EBPβ activation via PI3K/Akt and ERK1/2. Importantly, ISO-induced hypertrophy (increased cell surface area and protein and not DNA synthesis) was blunted in cardiomyocytes isolated from IL-18BP Tg mice, by the forced expression of IL-18BP, and IL-18 neutralizing Ab. Further, ISO treatment in vivo was associated with Akt and ERK1/2 phosphorylation, CREB and C/EBPβ activation, and IL-18BP expression. Notably, ISO-induced myocardial hypertrophy was markedly attenuated in IL-18BP Tg mice. These data provide the first evidence that ISO induces mouse IL-18BP expression in cardiomyocytes via β 2 -AR/Gi-mediated Akt and ERK1/2-dependent CREB and C/EBPβ activation. Moreover, IL-18BP inhibits ISO-induced cardiomyocyte hypertrophy both in vitro and in vivo , likely via prevention of the biologic effects of IL-18. Thus, catecholamine induced expression of IL-18BP may play a protective role in the hypertrophied failing heart, a disease state characterized by sustained β-AR activation and IL-18 signaling.

Abstract 17567: Stanniocalcin-1 Induces A Novel Anti-Oxidant Pathway in Cardiomyocytes

Rationale: We have shown increased cardiac stanniocalcin-1 (STC1) in patients with idiopathic dilated cardiomyopathy. STC1 localizes to the inner mitochondrial, and transgenic overexpression of STC1 is associated with increased energy utilization. Objective: We examined the hypothesis that STC1 uncouples mitochondrial oxidative phosphorylation - to affect energy metabolism and superoxide generation in cardiomyocytes. Methods and Results: Compared to WT mouse heart, STC1 Tg heart displays 2-fold higher uncoupling protein 3 (UCP3) levels, 40% lower ATP levels, but similar activities of respiratory chain complexes I-IV. In cultured adult rat cardiomyocytes, recombinant hSTC1 is internalized by cultured rat cardiomyocytes and is targeted to the mitochondria within minutes; it induces UCP3, decreases mitochondrial membrane potential and suppresses baseline and ANG II-provoked superoxide generation. In freshly-isolated adult mouse cardiomyocytes, STC1 diminishes baseline and ANG II-induced superoxide generation equally in WT and gp91 phox -/- cardiomyocytes, suggesting gp91phox-independent mechanism of action. Baseline superoxide generation is higher in freshly-isolated adult UCP3 -/- mouse cardiomyocytes, compared to WT; and, treatment of UCP3 -/- cardiomyocytes with STC1 failed to suppress superoxide generation in the presence or absence of ANG II, suggesting that the effects of STC1 on superoxide generation are UCP3-mediated. Conclusion: STC1 activates a novel anti-oxidant pathway in cardiac myocytes through induction of UCP3; this pathway may play an important role in regulating reactive oxygen species in the heart under normal physiologic and pathophysiologic conditions, and ameliorate the deleterious effects of ANG II-mediated cardiac injury.

Abstract 8420: Critical Role of CIKS/Act1 In Lipopolysaccharide (LPS)-Induced Cardiac Dysfunction i n vivo And Cardiomyocyte Contractility in vitro

The innate immune system and inflammatory cytokines are critical in the initiation and perpetuation of tissue injury. Toll-like receptor (TLR)4 recognizes bacterial LPS, and is currently known to signal via distinct MyD88-dependent and MyD88-independent (TRAM-dependent) pathways to activate NF-κB and AP-1, and induce proinflammatory cytokines such as interleukin (IL)-18. Since the adapter molecule CIKS (connection to IκB kinase and stress-activated protein kinases; also known as Act1 or traf3ip2) lies upstream of IKK and JNK, and since both TLR4 and CIKS contain TIR (Toll/Interleukin-1 receptor) domains , we hypothesized that CIKS can interact with TLR4, and contribute independently to LPS-induced contractile depression. Treatment with LPS inhibited adult mouse cardiomyocyte contractility by 54% (IonOptix), an effect partially reversed by MyD88 or TRAM knockdown. Significantly, CIKS knockdown also reversed LPS-induced contractile depression. Using GST pull-down and reciprocal immunoprecipitation/immunoblotting, we found that TLR4 can physically associate with CIKS, and this association is enhanced by LPS. LPS upregulates CIKS protein expression, and CIKS knockdown inhibited LPS-induced IKK/NF-κB and JNK/AP-1 activation, IL-18 expression, and contractile depression. LPS-mediated contractile depression was reversed by IL-18 neutralizing Ab and IL-18 binding protein (IL-18BP, a natural inhibitor of IL-18). These results were confirmed in cardiomyocytes isolated from TLR4-mutant, CIKS null, IL-18 null and IL-18BP Tg mice. Notably, systemic LPS administration reduced cardiac ejection fraction by 43% at 6 h in wild type mice, and this effect was markedly attenuated in TLR4-mutant, CIKS and IL-18 null, and IL-18BP Tg mice. These novel observations identify a TLR4/CIKS pathway in NF-κB and AP-1 activation in cardiomyocytes, and appears to be distinct from the well-characterized MyD88 and TRAM-dependent LPS/TLR4 signaling. These data also show that LPS induces contractile dysfunction in part via CIKS-mediated IKK/NF-κB and JNK/AP-1-dependent IL-18 expression. These data identify CIKS as a novel therapeutic target in conditions of innate immune activation and inflammation such as myocardial injury and congestive heart failure.

Tbx20 regulates a genetic program essential to adult mouse cardiomyocyte function

Human mutations in or variants of TBX20 are associated with congenital heart disease, cardiomyopathy, and arrhythmias. To investigate whether cardiac disease in patients with these conditions results from an embryonic or ongoing requirement for Tbx20 in myocardium, we ablated Tbx20 specifically in adult cardiomyocytes in mice. This ablation resulted in the onset of severe cardiomyopathy accompanied by arrhythmias, with death ensuing within 1 to 2 weeks of Tbx20 ablation. Accounting for this dramatic phenotype, we identified molecular signatures that posit Tbx20 as a central integrator of a genetic program that maintains cardiomyocyte function in the adult heart. Expression of a number of genes encoding critical transcription factors, ion channels, and cytoskeletal/myofibrillar proteins was downregulated consequent to loss of Tbx20. Genome-wide ChIP analysis of Tbx20-binding regions in the adult heart revealed that many of these genes were direct downstream targets of Tbx20 and uncovered a previously undescribed DNA-binding site for Tbx20. Bioinformatics and in vivo functional analyses revealed a cohort of transcription factors that, working with Tbx20, integrated multiple environmental signals to maintain ion channel gene expression in the adult heart. Our data provide insight into the mechanisms by which mutations in TBX20 cause adult heart disease in humans.

β2 adrenergic activation induces the expression of IL-18 binding protein, a potent inhibitor of isoproterenol induced cardiomyocyte hypertrophy in vitro and myocardial hypertrophy in vivo

Both the sympathetic nervous system and the proinflammatory cytokine interleukin-18 (IL-18) play key roles in the pathophysiology of the hypertrophied failing heart. IL-18 binding protein (IL-18BP), a natural inhibitor of IL-18, counters its biological effects. β-AR stimulation induces IL-18 expression, but whether it also regulates IL-18BP is not known. Here we demonstrate that the β-AR agonist isoproterenol (ISO) increases steady state IL-18BP mRNA and protein levels in adult mouse cardiomyocytes in a β2-AR-dependent manner. We cloned mouse Il18bp 5′cis-regulatory region, and identified putative CREB and C/EBPβ transcription factor-binding sites. Forced expression of mutant CREB or C/EBPβ knockdown markedly attenuated ISO-induced Il18bp transcription and deletion or mutation of CREB and C/EBP motifs in the Il18bp promoter reduced ISO-induced promoter-reporter gene activity. ISO induced CREB and C/EBPβ activation in cardiomyocytes via PI3K/Akt and ERK1/2. Importantly, ISO-induced hypertrophy in vitro was dependent on IL-18 induction as it was blunted by IL-18 neutralizing antibodies and forced expression of IL-18BP. Moreover, ISO-induced hypertrophy was markedly attenuated in IL-18 null and IL-18BP transgenic mice. These data support the novel concept that β-AR activation, in addition to inducing cardiomyocyte hypertrophy via IL-18, concomitantly induces a countering effect by stimulating IL-18BP expression, and that ISO-induced cardiomyocyte hypertrophy may result from a net effect of IL-18 and IL-18BP induction.

Calpain activation contributes to myocardial apoptosis induced by ischemia-reperfusion

BackgroundCalpain is a family of cytoplasmic cysteine proteases activated by calcium ions. The mechanistic link between apoptosis and activation of calpain remains unclear. Apoptotic mechanism has been implicated in myocardial...

Calpain activation contributes to myocardial apoptosis induced by ischemia-reperfusion

BackgroundCalpain is a family of cytoplasmic cysteine proteases activated by calcium ions. The mechanistic link between apoptosis and activation of calpain remains unclear. Apoptotic mechanism have been implicated in myocardial...

Calpain activation contributes to adult mouse cardiomyocytes injury induced by H2O2

BackgroundAlthough restoration of blood flow after ischemia is essential for cardiomyocytes salvation, reperfusion itself exacerbates myocardial injury. Considerable reactive oxygen species (ROS) are produced by the myocardium and infiltrating inflammatory...

CaMKIIδB Mediates Aberrant NCX1 Expression and the Imbalance of NCX1/SERCA in Transverse Aortic Constriction-Induced Failing Heart

Ca2+/calmodulin-dependent protein kinase II δB (CaMKIIδB) is one of the predominant isoforms of CaMKII in the heart. The precise role of CaMKIIδB in the transcriptional cross-talk of Ca2+-handling proteins during heart failure remains unclear. In this work, we aim to determine the mechanism of CaMKIIδB in modulating the expression of sarcolemmal Na+–Ca2+ exchange (NCX1). We also aim to address the potential effects of calmodulin antagonism on the imbalance of NCX1 and sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) during heart failure. Eight weeks after transverse aortic constriction (TAC)-induced heart failure in mice, we found that the heart weight/tibia length (HW/TL) ratio and the lung weight/body weight (LW/BW) ratio increased by 59% and 133%, respectively. We further found that the left ventricle-shortening fraction decreased by 40% compared with the sham-operated controls. Immunoblotting revealed that the phosphorylation of CaMKIIδB significantly increased 8 weeks after TAC-induced heart failure. NCX1 protein levels were also elevated, whereas SERCA2 protein levels decreased in the same animal model. Moreover, transfection of active CaMKIIδB significantly increased NCX1 protein levels in adult mouse cardiomyocytes via class IIa histone deacetylase (HDAC)/myocyte enhancer factor-2 (MEF2)-dependent signaling. In addition, pharmacological inhibition of calmodulin/CaMKIIδB activity improved cardiac function in TAC mice, which partially normalized the imbalance between NCX1 and SERCA2. These data identify NCX1 as a cellular target for CaMKIIδB. We also suggest that the CaMKIIδB-induced imbalance between NCX1 and SERCA2 is partially responsible for the disturbance of intracellular Ca2+ homeostasis and the pathological process of heart failure.

Metformin protects against doxorubicin-induced cardiotoxicity: Involvement of the adiponectin cardiac system

Doxorubicin has cardiotoxic effects that limit its clinical benefit in cancer patients. Metformin exerts cardioprotective actions via AMP-activated protein kinase (AMPK) and increases the expression of adiponectin and its receptors (adipoR1 and adipoR2) in skeletal muscle and adipose tissue, but its effect on cardiac tissue is still unknown. This work aimed to study whether metformin exerts any protective action against the cardiotoxicity of doxorubicin and whether the cardiac system of adiponectin is involved in any such action. The addition of doxorubicin (5μM) to adult mouse cardiomyocytes (HL-1 cell line) induced apoptosis, which was characterized by a loss of cell viability, activation of caspases, and fragmentation of the genetic material. Doxorubicin treatment also caused a decrease in the activity of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase. Pretreatment with metformin (4mM, 24h) provided protection against doxorubicin-induced damage. This pretreatment significantly increased cell viability, attenuated the activation of caspases and the fragmentation of genetic material, and restored the antioxidant activity. In addition, metformin up-regulated the expression of adiponectin and its receptors, adipoR1 and adipoR2, in cardiomyocytes. In contrast, silencing either adipoR1 or adipoR2 with siRNA inhibited the AMPK activation and the protective effects of metformin. Taken together, these results demonstrate that metformin protects cardiomyocytes from doxorubicin-induced damage and that the cardiac adiponectin system plays an important role in this protective action.

74 Mechanistic Study for the role of advanced glycation end products in the development of diabetic heart failure

Advanced glycation end products (AGEs) are thought to play a crucial role in the development of diabetic complications including heart failure, a leading cause of morbidity and mortality in diabetic...

A method to fix and permeabilize isolated adult mouse cardiomyocytes for immuno-staining and confocal imaging

A method to fix and permeabilize isolated adult mouse cardiomyocytes for immuno-staining and confocal imaging

Acrolein, an Environmental Toxin, Induces Cardiomyocyte Apoptosis via Elevated Intracellular Calcium and Free Radicals

Acrolein, an unsaturated aldehyde, is an environmental toxin known to inhibit mitochondrial electron transport chain in brain and induce lipid peroxidation and apoptosis. However, the nature of the effects of acrolein on cardiac function and myocardium is not known. The objective of this study is to examine whether acrolein induces apoptosis in cardiomyocytes and alters cytosolic calcium concentration and the intracellular oxygen free-radical levels. Adult mouse cardiomyocytes exposed to 1μmol/l of acrolein showed a marked increase in the intracellular oxygen free-radicals and calcium concentration, by 12- and 2-fold, respectively, compared to the resting value. Moreover, the cardiomyocyte viability decreased significantly in a dose-dependent manner by treatment with 25, 50, and 100μmol/l of acrolein compared to controls. Morphological changes and DNA laddering typical of apoptosis were found in acrolein-exposed cardiomyocytes. Our finding suggested that acrolein caused apoptotic death of adult mice cardiomyocytes by increasing intracellular oxygen free-radicals and calcium concentration.

The coxsackie–adenovirus receptor induces an inflammatory cardiomyopathy independent of viral infection

The coxsackie–adenovirus receptor (CAR) is a viral receptor for Group B coxsackieviruses (CVBs) and adenoviruses. CAR has been linked with the innate immune response to CVB myocarditis, and with activation of inflammatory cells in vitro. We hypothesized that CAR activates signals that promote inflammation in the myocardium independent of viral infection. To test this we conditionally overexpressed murine CAR in cardiomyocytes of adult binary transgenic mice under the control of a tetracycline-responsive (tet-off) α-myosin heavy chain (αMtTA) promoter (mCAR +/αMtTA + mice). An inflammatory cardiomyopathy developed in both lines generated (6-mCAR +/αMtTA + and 12-mCAR +/αMtTA +) following withdrawal of doxycycline. Cardiac CAR was upregulated at 4 weeks of age in 6-mCAR +/αMtTA + mice and induced a mild inflammatory infiltrate (score 1.3 of 4.0 ± 0.3) at 6 weeks, with 95% of mice surviving to that time. In the second line, 12-mCAR +/αMtTA + mice, CAR was upregulated in the majority of mice by 3 weeks of age, and by 5 weeks of age more severe cardiac inflammation (score 2.8 of 4.0 ± 0.4) developed with only 56% of mice surviving. The cardiac inflammatory infiltrate was primarily natural killer cells and macrophages in both mCAR +/αMtTA + lines. A proinflammatory cytokine response with increased cardiac interferon-γ, interleukin (IL)-12, IL-1β, tumor necrosis factor-α and IL-6 was detected by real-time RT-PCR. CAR has been linked to signaling via the inflammatory mitogen-activated protein kinase (MAPK) cascades; therefore, we evaluated the response of these pathways in hearts with upregulated CAR. Both stress-activated JNK and p38MAPK were activated in mCAR +/αMtTA + hearts prior to onset of inflammation and in isolated mCAR +/αMtTA + cardiomyocytes. In conclusion, we show for the first time that CAR upregulation in the adult mouse heart induces cardiac inflammation reminiscent of early viral myocarditis. CAR-induced stress-activated MAPK signaling may contribute to the development of cardiac inflammation unrelated to viral infection per se.

Cardiomyocyte marker expression in a human lymphocyte cell line using mouse cardiomyocyte extract

Cell transplantation shows potential for the treatment of cardiac diseases. Embryonic stem cells, cord blood and mesenchymal stem cells have been suggested as sources for transplantation therapy. Because of some technical limitations with the use of stem cells, transdifferentiation of fully differentiated cells is a potentially useful alternative. We investigated whether human peripheral blood cells could transdifferentiate into cardiomyocyte. Transdifferentiation was induced in a human B lymphocyte cell line (Raji). Cardiomyocyte extract was prepared from adult mouse cardiomyocytes. The cells were treated with 5-aza-2-deoxycytidine and trichostatin A, permeabilized with streptolysin O, and exposed to the mouse cardiomyocyte extract. They were cultured for 10 days, 3 weeks and 4 weeks. Cardiomyocyte markers were detected with immunohistochemistry and flow cytometry. Immunocytochemistry revealed that some cells expressed myosin heavy chain, α-actinin and cardiac troponin T after 3 and 4 weeks. Flow cytometry confirmed these data. In cells exposed to trichostatin A and 5-aza-2-deoxycytidine and permeabilized in the presence of the cardiomyocyte extract, troponin T expression was seen in 3.53% of the cells and 3.11% of them expressed α-actinin. After exposure to the cardiomyocyte extract, some permeabilized cells adhered to the plate loosely; however, the morphology did not change significantly, and they continued to show a rounded shape after 4 weeks. Our treated lymphocytes expressed cardiomyocyte markers. Our results suggest that lymphocytes may be useful in future research as a source of cells for reprogramming procedures.

An Improved Isolation Procedure for Adult Mouse Cardiomyocytes

Isolated adult mouse cardiomyocytes are an important tool in cardiovascular research, but are challenging to prepare. Because the energy supply determines cell function and viability, we compared total creatine ([Cr]) and [ATP] in isolated cardiomyocytes with the intact mouse heart. Isolated myocytes suffered severe losses of Cr (-70%) and ATP (-53%). Myocytes were not able to replete [Cr] during a 5h incubation period in medium supplemented with 1mM Cr. In contrast, adding 20mM Cr to the digestion buffers was sufficient to maintain normal [Cr]. Supplementing buffers with 5mM of inosine (Ino) and adenosine (Ado) to prevent loss of cellular nucleosides partially protected against loss of ATP. To test whether maintaining [ATP] and [Cr] improves contractile function, myocytes were challenged by varying pacing rate from 0.5 to 10Hz and by adding isoproterenol (Iso) at 5 and 10Hz. All groups performed well up to 5Hz, showing a positive cell shortening-frequency relationship; however, only 16% of myocytes isolated under standard conditions were able to sustain pacing with Iso challenge at 10Hz. In contrast, 30-50% of the myocytes with normal Cr levels were able to contract and maintain low diastolic [Ca(2+)]. Cell yield also improved in Cr and the Cr/Ino/Ado-treated groups (85-90% vs. 70-75% rod shaped in untreated myocytes). These data suggest that viability and performance of isolated myocytes are improved when they are protected from the severe loss of Cr and ATP during the isolation, making them an even better research tool.

Occult Myopathic Effects of K118c Mutation in Cardiac Troponin I Detected in Isolated and Cultured Adult Mouse Cardiomyocyte

Isolated adult cardiomyocytes provide a valuable system for cellular level phenotype studies. However, an N-terminal truncation of cardiac troponin T (cTnT-ND) found in ischemia-reperfusion with functional consequences also occurs during the perfusion-based cell isolation. Here we utilized short-term culture to allow isolated cardiomyocytes to recover for the study of a cardiac troponin I mutation (cTnI-K118C). Adult cardiomyocytes were isolated from wild type and cTnl-K118C transgenic mice. Myofilament proteins were monitored using SDS-PAGE, Western blot and Pro-Q phosphoprotein staining. Paced contraction was examined on day 0 and day 2 of culture using edge detection. The cultured adult mouse cardiomyocytes maintained normal contents of myosin, tropomyosin, cTnT and cTnI in culture for over 4 days. cTnT-ND declined from ∼7.7% of total cTnT at day 0 to ∼2.9% after 2 days of culture. The PKA-dependent phosphorylation of cTnI was preserved in 2 day-cultured cells. The amplitude of shortening in wild type myocytes on day 0 and day 2 were 3.05±0.53% and 3.79±0.68%, respectively, without statistical difference. The amplitude of shortening and the velocities of shortening/re-lengthening were decreased in day 0 and 2-day-cultured cTnI-K118C cardiomyocytes as compared with wild type controls, but only the 2-day-cultured group showed statistical significance (P<0.05). The slack length of the cardiomyocytes was 119.4±2.0 µm for wild type and 146.1±2.9 µm for cTnI-K118C groups (P<0.01) with no increase in sarcomere length or cell diameter. The data demonstrated that 2-day-cultured adult mouse cardiomyocytes were effectively recovering from the proteolytic modification of cTnT induced during isolation while maintaining the original level of contractility. The study of isolated cardiomyocytes from young adult transgenic mice without heart failure revealed that cTnI-118C decreased contractility and caused cellular remodeling toward dilated cardiomyopathy.

FGF13 is a Regulator of the Cardiac Voltage-Gated Sodium Channel Nav1.5

The four members of the intracellular Fibroblast Growth Factor (iFGF) subfamily, FGF11-14, regulate voltage-gated sodium (Nav) channels. A missense mutation in FGF14 causes spinocerebellar ataxia 27, thought to be due to Nav channel dysfunction. iFGFs are expressed in the heart, but whether they regulate cardiac Nav channels is not known. Using quantitative real-time RT-PCR, we identified that FGF13 isoforms are the dominant iFGFs in adult mouse heart. Using whole cell patch-clamp configuration, we determined a functional link between FGF13 and Nav1.5. We found that the FGF13 isoforms, FGF13S, FGF13U, and FGF13VY differentially modulate Nav1.5 current density when transiently co-expressed in HEK293 cells. Steady-state activation was not altered. In contrast, steady-state availability was significantly shifted towards the depolarizing direction by each of the FGF13 isoforms. Most strikingly, FGF13S induced a dramatic slowing of recovery from inactivation. Co-immunoprecipitation showed that FGF13 interacted with Nav1.5 in cardiomyocytes. Using a pull down assay, we found that FGF13 directly interacted with C-terminus of Nav1.5. Immunostaining showed that FGF13 co-localized with Nav1.5 on sarcolemma. Some FGF13 is present in subcellular regions devoid of Nav1.5, suggesting other roles than sodium channel modulation. FGF13 knockdown by adenoviral infection with shRNA in adult mice cardiomyocytes affected sodium current density and steady-state availability. FGF13 knockdown in a neonatal rat cardiomyocyte monolayer reduced cardiac impulse conduction and the velocity of the action potential upstroke. These data are the first report of FGF13 as regulator of Nav1.5 in heart and suggest that FGF13 isoform-specific regulation of cardiac Nav channels plays important physiological and pathophysiological roles.