Wild-type Cardiomyocytes Research Articles

Characterization of CaV1.2 exon 33 heterozygous knockout mice and negative correlation between Rbfox1 and CaV1.2 exon 33 expressions in human heart failure

ABSTRACTRecently, we reported that homozygous deletion of alternative exon 33 of CaV1.2 calcium channel in the mouse resulted in ventricular arrhythmias arising from increased CaV1.2Δ33 ICaL current density in the cardiomyocytes. We wondered whether heterozygous deletion of exon 33 might produce cardiac phenotype in a dose-dependent manner, and whether the expression levels of RNA splicing factors known to regulate alternative splicing of exon 33 might change in human heart failure. Unexpectedly, we found that exon 33+/− cardiomyocytes showed similar CaV1.2 channel properties as wild-type cardiomyocyte, even though CaV1.2Δ33 channels exhibit a gain-in-function. In human hearts, we found that the mRNA level of splicing factor Rbfox1, but not Rbfox2, was downregulated in dilated cardiomyopathy, and CACNA1C mRNA level was dramatically decreased in the both of dilated and ischemic cardiomyopathy. These data imply Rbfox1 may be involved in the development of cardiomyopathies via regulating the alternative splicing of CaV1.2 exon 33. (149 words)

The Effects of Aging on the Regulation of T-Tubular ICa by Caveolin in Mouse Ventricular Myocytes.

Aging is associated with diminished cardiac function in males. Cardiac excitation-contraction coupling in ventricular myocytes involves Ca influx via the Ca current (ICa) and Ca release from the sarcoplasmic reticulum, which occur predominantly at t-tubules. Caveolin-3 regulates t-tubular ICa, partly through protein kinase A (PKA), and both ICa and caveolin-3 decrease with age. We therefore investigated ICa and t-tubule structure and function in cardiomyocytes from male wild-type (WT) and caveolin-3-overexpressing (Cav-3OE) mice at 3 and 24 months of age. In WT cardiomyocytes, t-tubular ICa-density was reduced by ~50% with age while surface ICa density was unchanged. Although regulation by PKA was unaffected by age, inhibition of caveolin-3-binding reduced t-tubular ICa at 3 months, but not at 24 months. While Cav-3OE increased cardiac caveolin-3 protein expression ~2.5-fold at both ages, the age-dependent reduction in caveolin-3 (WT ~35%) was preserved in transgenic mice. Overexpression of caveolin-3 reduced t-tubular ICa density at 3 months but prevented further ICa loss with age. Measurement of Ca release at the t-tubules revealed that the triggering of local Ca release by t-tubular ICa was unaffected by age. In conclusion, the data suggest that the reduction in ICa density with age is associated with the loss of a caveolin-3-dependent mechanism that augments t-tubular ICa density.

Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts.

Cardiac insulin resistance is a key pathogenic factor for diabetic cardiomyopathy (DCM), but the mechanism remains largely unclear. We found that diabetic hearts exhibited decreased phosphorylation of total Akt and isoform Akt2 but not Akt1 in wild-type (WT) male FVB mice, which was accompanied by attenuation of Akt downstream glucose metabolic signal. All of these signal changes were not observed in metallothionein cardiac-specific transgenic (MT-TG) hearts. Furthermore, insulin-induced glucose metabolic signals were attenuated only in WT diabetic hearts. In addition, diabetic hearts exhibited increased Akt-negative regulator tribbles pseudokinase 3 (TRB3) expression only in WT mice, suggesting that MT may preserve Akt2 function via inhibiting TRB3. Moreover, MT prevented tert-butyl hydroperoxide (tBHP)-reduced insulin-stimulated Akt2 phosphorylation in MT-TG cardiomyocytes, which was abolished by specific silencing of Akt2. Specific silencing of TRB3 blocked tBHP inhibition of insulin-stimulated Akt2 phosphorylation in WT cardiomyocytes, whereas overexpression of TRB3 in MT-TG cardiomyocytes and hearts abolished MT preservation of insulin-stimulated Akt2 signals and MT prevention of DCM. Most importantly, supplementation of Zn to induce MT preserved cardiac Akt2 signals and prevented DCM. These results suggest that diabetes-inhibited cardiac Akt2 function via TRB3 upregulation leads to aberrant cardiac glucose metabolism. MT preservation of cardiac Akt2 function by inhibition of TRB3 prevents DCM.

Mdm2 regulates cardiac contractility by inhibiting GRK2-mediated desensitization of β-adrenergic receptor signaling.

The oncoprotein Mdm2 is a RING domain-containing E3 ubiquitin ligase that ubiquitinates G protein-coupled receptor kinase 2 (GRK2) and β-arrestin2, thereby regulating β-adrenergic receptor (βAR) signaling and endocytosis. Previous studies showed that cardiac Mdm2 expression is critical for controlling p53-dependent apoptosis during early embryonic development, but the role of Mdm2 in the developed adult heart is unknown. We aimed to identify if Mdm2 affects βAR signaling and cardiac function in adult mice. Using Mdm2/p53-KO mice, which survive for 9-12 months, we identified a critical and potentially novel role for Mdm2 in the adult mouse heart through its regulation of cardiac β1AR signaling. While baseline cardiac function was mostly similar in both Mdm2/p53-KO and wild-type (WT) mice, isoproterenol-induced cardiac contractility in Mdm2/p53-KO was significantly blunted compared with WT mice. Isoproterenol increased cAMP in left ventricles of WT but not of Mdm2/p53-KO mice. Additionally, while basal and forskolin-induced calcium handling in isolated Mdm2/p53-KO and WT cardiomyocytes were equivalent, isoproterenol-induced calcium handling in Mdm2/p53-KO was impaired. Mdm2/p53-KO hearts expressed 2-fold more GRK2 than WT. GRK2 polyubiquitination via lysine-48 linkages was significantly reduced in Mdm2/p53-KO hearts. Tamoxifen-inducible cardiomyocyte-specific deletion of Mdm2 in adult mice also led to a significant increase in GRK2, and resulted in severely impaired cardiac function, high mortality, and no detectable βAR responsiveness. Gene delivery of either Mdm2 or GRK2-CT in vivo using adeno-associated virus 9 (AAV9) effectively rescued β1AR-induced cardiac contractility in Mdm2/p53-KO. These findings reveal a critical p53-independent physiological role of Mdm2 in adult hearts, namely, regulation of GRK2-mediated desensitization of βAR signaling.

The Calcineurin-FoxO-MuRF1 signaling pathway regulates myofibril integrity in cardiomyocytes.

Altered Ca2+ handling is often present in diseased hearts undergoing structural remodeling and functional deterioration. However, whether Ca2+ directly regulates sarcomere structure has remained elusive. Using a zebrafish ncx1 mutant, we explored the impacts of impaired Ca2+ homeostasis on myofibril integrity. We found that the E3 ubiquitin ligase murf1 is upregulated in ncx1-deficient hearts. Intriguingly, knocking down murf1 activity or inhibiting proteasome activity preserved myofibril integrity, revealing a MuRF1-mediated proteasome degradation mechanism that is activated in response to abnormal Ca2+ homeostasis. Furthermore, we detected an accumulation of the murf1 regulator FoxO in the nuclei of ncx1-deficient cardiomyocytes. Overexpression of FoxO in wild type cardiomyocytes induced murf1 expression and caused myofibril disarray, whereas inhibiting Calcineurin activity attenuated FoxO-mediated murf1 expression and protected sarcomeres from degradation in ncx1-deficient hearts. Together, our findings reveal a novel mechanism by which Ca2+ overload disrupts myofibril integrity by activating a Calcineurin-FoxO-MuRF1-proteosome signaling pathway.

Myeloperoxidase Mediates Postischemic Arrhythmogenic Ventricular Remodeling.

Ventricular arrhythmias remain the leading cause of death in patients suffering myocardial ischemia. Myeloperoxidase, a heme enzyme released by polymorphonuclear neutrophils, accumulates within ischemic myocardium and has been linked to adverse left ventricular remodeling. To reveal the role of myeloperoxidase for the development of ventricular arrhythmias. In different murine models of myocardial ischemia, myeloperoxidase deficiency profoundly decreased vulnerability for ventricular tachycardia on programmed right ventricular and burst stimulation and spontaneously as assessed by ECG telemetry after isoproterenol injection. Experiments using CD11b/CD18 integrin-deficient (CD11b-/-) mice and intravenous myeloperoxidase infusion revealed that neutrophil infiltration is a prerequisite for myocardial myeloperoxidase accumulation. Ventricles from myeloperoxidase-deficient (Mpo-/-) mice showed less pronounced slowing and decreased heterogeneity of electric conduction in the peri-infarct zone than wild-type mice. Expression of the redox-sensitive gap junctional protein Cx43 (Connexin 43) was reduced in the peri-infarct area of wild-type compared with Mpo-/- mice. In isolated wild-type cardiomyocytes, Cx43 protein content decreased on myeloperoxidase/H2O2 incubation. Mapping of induced pluripotent stem cell-derived cardiomyocyte networks and in vivo investigations linked Cx43 breakdown to myeloperoxidase-dependent activation of matrix metalloproteinase 7. Moreover, Mpo-/- mice showed decreased ventricular postischemic fibrosis reflecting reduced accumulation of myofibroblasts. Ex vivo, myeloperoxidase was demonstrated to induce fibroblast-to-myofibroblast transdifferentiation by activation of p38 mitogen-activated protein kinases resulting in upregulated collagen generation. In support of our experimental findings, baseline myeloperoxidase plasma levels were independently associated with a history of ventricular arrhythmias, sudden cardiac death, or implantable cardioverter-defibrillator implantation in a cohort of 2622 stable patients with an ejection fraction >35% undergoing elective diagnostic cardiac evaluation. Myeloperoxidase emerges as a crucial mediator of postischemic myocardial remodeling and may evolve as a novel pharmacological target for secondary disease prevention after myocardial ischemia.

Diltiazem prevents stress-induced contractile deficits in cardiomyocytes, but does not reverse the cardiomyopathy phenotype in Mybpc3-knock-in mice.

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac illness and can lead to diastolic dysfunction, sudden cardiac death and heart failure. Treatment of HCM patients is empirical and current pharmacological treatments are unable to stop disease progression or reverse hypertrophy. In this study, we tested if the non-dihydropyridine Ca2+ channel blocker diltiazem, which previously showed potential to stop disease progression, can improve the phenotype of a HCM mouse model (Mybpc3-targeted knock-in), which is based on a mutation commonly found in patients. Diltiazem improved contractile function of isolated ventricular cardiomyocytes acutely, but chronic application did not improve the phenotype of adult mice with a fully developed HCM. Our study shows that diltiazem has beneficial effects in HCM, but long-term treatment success is likely to depend on characteristics and cause of HCM and onset of treatment. Left ventricular hypertrophy, diastolic dysfunction and fibrosis are the main features of hypertrophic cardiomyopathy (HCM). Guidelines recommend β-adrenoceptor or Ca2+ channel antagonists as pharmacological treatment. The Ca2+ channel blocker diltiazem recently showed promising beneficial effects in pre-clinical HCM, particularly in patients carrying MYBPC3 mutations. In the present study we evaluated whether diltiazem could ameliorate or reverse the disease phenotype in cells and in vivo in an Mybpc3-targeted knock-in (KI) mouse model of HCM. Sarcomere shortening and Ca2+ transients were measured in KI and wild-type (WT) cardiomyocytes in basal conditions (1-Hz pacing) and under stress conditions (30nm isoprenaline, 5-Hz pacing) with or without pre-treatment with 1μm diltiazem. KI cardiomyocytes exhibited lower diastolic sarcomere length (dSL) at baseline, a tendency to a stronger positive inotropic response to isoprenaline than WT, a marked reduction of dSL and a tendency towards arrhythmias under stress conditions. Pre-treatment of cardiomyocytes with 1μm diltiazem reduced the drop in dSL and arrhythmia frequency in KI, and attenuated the positive inotropic effect of isoprenaline. Furthermore, diltiazem reduced the contraction amplitude at 5Hz but did not affect diastolic Ca2+ load and Ca2+ transient amplitude. Sixmonths of diltiazem treatment of KI mice did not reverse cardiac hypertrophy and dysfunction, activation of the fetal gene program or fibrosis. In conclusion, diltiazem blunted the response to isoprenaline in WT and KI cardiomyocytes and improved diastolic relaxation under stress conditions in KI cardiomyocytes. This beneficial effect of diltiazem in cells did not translate in therapeutic efficacy when applied chronically in KI mice.

Impact of cGMP-PKG Pathway Modulation on Titin Phosphorylation and Titin-Based Myocardial Passive Stiffness

RATIONALE: The crucial contribution of the giant myofilament protein titin to diastolic stiffness and cardiomyocyte passive force (Fpassive) is dependent, in part, on titin isoform composition and phosphorylation. Phosphorylation of titin by cyclic guanosine monophosphate (cGMP)-dependent protein kinase G (PKG) lowers titin-based stiffness, thus mediating a mechanical signaling process that is disturbed in heart failure. OBJECTIVE: To elucidate which elements of the nitric oxide (NO) cGMP-PKG signaling network are critical for titin phosphorylation and stiffness in vivo. METHODS AND RESULTS: We employed genetic knockout (KO) mouse models deficient for enzymes of the cGMP-PKG pathway, including cardiomyocyte-specific deletion of the guanylyl cyclase (GC)-A receptor and cGMP-dependent PKG (cGKI), as well as global deletion of soluble GC (sGC). We assessed titin phosphorylation by immunoblotting using phosphoserine-specific titin antibodies and by mass spectrometry quantification using stable isotope labeling of amino acids in mixed cultures of heart tissue from either wild-type (WT) or KO mice. The Fpassive of single permeabilized cardiomyocytes was recorded before and after administration of PKG. In all three genetic models, all-titin phosphorylation was reduced compared to WT hearts. The important PKG-dependent phospho-S4080 site within the N2-Bus region of mouse titin was hypophosphorylated in all three KO models. Unexpectedly, mass spectrometry analysis revealed that most class 1 titin phospho-sites within the molecular spring segment, including the Ig-domain regions, were hyperphosphorylated. Only a few sites showed a phosphorylation deficit or remaining unchanged. Particularly in the cGKI model many class 1 phospho-sites were hyperphosphorylated compared to WT hearts, indicative of the presence of compensatory processes following loss of PKG; indeed, this was associated with upregulation of several kinases that phosphorylate titin, including CaMKII, ERK2 and PKCα and with a clear rise in Fpassive in KO vs. WT cardiomyocytes. We also found an upregulation of phosphodiesterase 3, but downregulation of phosphodiesterase 9a, which regulates natriuretic peptides rather than nitric oxide-stimulated cGMP in heart myocytes. While administration of PKG lowered Fpassive of WT and KO cardiomyocytes in all models, this effect was more pronounced in the cGKI KO. CONCLUSIONS: Multiple in vivo phosphorylated class I titin phospho-sites were identified within the molecular spring segment, some of which depended on the cGMP-PKG pathway. While cGMP-activated PKG remains an important titin-targeting kinase, many titin phospho-sites may be regulated through a network of protein kinases/phosphatases.

Functional Effects of the RyR2R420Q Catecholaminergic Ventricular Polymorphic Tachycardia in Mouse Cardiomyocytes

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a genetic disease characterized by stress-induced syncope and/or sudden death. CPVT1 mutations are clustered in three regions: the N-terminal, the central, and the C-terminal domains of the cardiac Ca2+ release channel (type-2 ryanodine receptor, RyR2). We have created a knock-in (KI) mouse model bearing the RyR2R420Q mutation, which is located in the N-terminal region, found in three families diagnosed with CPVT. Electrocardiographic measurements of wake animals showed bidirectional ventricular tachycardia after epinephrine/caffeine injection in all the KI (heterozygous for RyR2R420Q) but in none of the WT (wild type) animals, validating the model. Consistent with the in-vivo phenotype, during action potential (AP) measurement by patch-clamp in the presence of 100 nM isoproterenol all KI myocytes displayed delayed afterdepolarizations (DADs) and triggered activity (TA), while the incidence of pro-arrhythmic events was significantly lower in WT myocytes. AP characteristics and potassium currents showed only minor differences between KI and WT. We measured ICa, [Ca2+]i transients and intra-SR [Ca2+] by patch-clamp and confocal microscopy. ICa density was similar between WT and KI cardiomyocytes, but the associated [Ca2+]i transient amplitude was unchanged or enhanced depending on the protocol, as well as its response to beta-adrenergic stimulation. Intact cardiac myocytes showed more Ca2+ sparks occurrence with a lowered SR Ca2+ threshold to initiate Ca2+ waves. Electron microscopy analyses indicate that Ca2+ release sites (or estimates indicate that couplons) in KI cardiomyocytes were fewer in number, significantly smaller and, hence, may contain a significantly lower number of RyR2. This morphological changes in couplons could participate to the differences in Ca2+ sparks characteristics recorded in KI cells compared to WT. Taken together, we have found a unique cellular alteration in this N terminal RyR2 mutation at structural and functional levels.

Absence of suppressor of cytokine signaling 2 turns cardiomyocytes unresponsive to LIF-dependent increases in Ca2+ levels.

Little is known regarding the role of suppressor of cytokine signaling (SOCS) in the control of cytokine signaling in cardiomyocytes. We investigated the consequences of SOCS2 ablation for leukemia inhibitory factor (LIF)-induced enhancement of intracellular Ca2+ ([Ca2+]i) transient by performing experiments with cardiomyocytes from SOCS2-knockout (ko) mice. Similar levels of SOCS3 transcripts were seen in cardiomyocytes from wild-type and SOCS2-ko mice, while SOCS1 mRNA was reduced in SOCS2-ko. Immunoprecipitation experiments showed increased SOCS3 association with gp130 receptor in SOCS2-ko myocytes. Measurements of Ca2+ in wild-type myocytes exposed to LIF showed a significant increase in the magnitude of the Ca2+ transient. This change was absent in LIF-treated SOCS2-ko cells. LIF activation of ERK and STAT3 was observed in both wild-type and SOCS2-ko cells, indicating that in SOCS2-ko, LIF receptors were functional, despite the lack of effect in the Ca2+ transient. In wild-type cells, LIF-induced increase in [Ca2+]i and phospholamban Thr17 [PLN(Thr17)] phosphorylation was inhibited by KN-93, indicating a role for CaMKII in LIF-induced Ca2+ raise. LIF-induced phosphorylation of PLN(Thr17) was abrogated in SOCS2-ko myocytes. In wild-type cardiomyocytes, LIF treatment increased L-type Ca2+ current (ICa,L), a key activator of CaMKII in response to LIF. Conversely, SOCS2-ko myocytes failed to activate ICa,L in response to LIF, providing a rationale for the lack of LIF effect on Ca2+ transient. Our data show that absence of SOCS2 turns cardiomyocytes unresponsive to LIF-induced [Ca2+] raise, indicating that endogenous levels of SOCS2 are crucial for full activation of LIF signaling in the heart.

Kvβ1.1 (AKR6A8) senses pyridine nucleotide changes in the mouse heart and modulates cardiac electrical activity.

The present study investigates the physiological role of Kvβ1 subunit for sensing pyridine nucleotide (NADH/NAD+) changes in the heart. We used Kvβ1.1 knockout (KO) or wild-type (WT) mice and established that Kvβ1.1 preferentially binds with Kv4.2 and senses the pyridine nucleotide changes in the heart. The cellular action potential duration (APD) obtained from WT cardiomyocytes showed longer APDs with lactate perfusion, which increases intracellular NADH levels, while the APDs remained unaltered in the Kvβ1.1 KO. Ex vivo monophasic action potentials showed a similar response, in which the APDs were prolonged in WT mouse hearts with lactate perfusion; however, the Kvβ1.1 KO mouse hearts did not show APD changes upon lactate perfusion. COS-7 cells coexpressing Kv4.2 and Kvβ1.1 were used for whole cell patch-clamp recordings to evaluate changes caused by NADH (lactate). These data reveal that Kvβ1.1 is required in the mediated inactivation of Kv4.2 currents, when NADH (lactate) levels are increased. In vivo, isoproterenol infusion led to increased NADH in the heart along with QTc prolongation in wild-type mice; regardless of the approach, our data show that Kvβ1.1 recognizes NADH changes and modulates Kv4.2 currents affecting AP and QTc durations. Overall, this study uses multiple levels of investigation, including the heterologous overexpression system, cardiomyocyte, ex vivo, and ECG, and clearly depicts that Kvβ1.1 is an obligatory sensor of NADH/NAD changes in vivo, with a physiological role in the heart.NEW & NOTEWORTHY Cardiac electrical activity is mediated by ion channels, and Kv4.2 plays a significant role, along with its binding partner, the Kvβ1.1 subunit. In the present study, we identify Kvβ1.1 as a sensor of pyridine nucleotide changes and as a modulator of Kv4.2 gating, action potential duration, and ECG in the mouse heart.

Epigallocatechin-3-Gallate Accelerates Relaxation and Ca2+ Transient Decay and Desensitizes Myofilaments in Healthy and Mybpc3-Targeted Knock-in Cardiomyopathic Mice.

Background: Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac muscle disease with left ventricular hypertrophy, interstitial fibrosis and diastolic dysfunction. Increased myofilament Ca2+ sensitivity could be the underlying cause of diastolic dysfunction. Epigallocatechin-3-gallate (EGCg), a catechin found in green tea, has been reported to decrease myofilament Ca2+ sensitivity in HCM models with troponin mutations. However, whether this is also the case for HCM-associated thick filament mutations is not known. Therefore, we evaluated whether EGCg affects the behavior of cardiomyocytes and myofilaments of an HCM mouse model carrying a gene mutation in cardiac myosin-binding protein C and exhibiting both increased myofilament Ca2+ sensitivity and diastolic dysfunction.Methods and Results: Acute effects of EGCg were tested on fractional sarcomere shortening and Ca2+ transients in intact ventricular myocytes and on force-Ca2+ relationship of skinned ventricular muscle strips isolated from Mybpc3-targeted knock-in (KI) and wild-type (WT) mice. Fractional sarcomere shortening and Ca2+ transients were analyzed at 37°C under 1-Hz pacing in the absence or presence of EGCg (1.8 μM). At baseline and in the absence of Fura-2, KI cardiomyocytes displayed lower diastolic sarcomere length, higher fractional sarcomere shortening, longer time to peak shortening and time to 50% relengthening than WT cardiomyocytes. In WT and KI neither diastolic sarcomere length nor fractional sarcomere shortening were influenced by EGCg treatment, but relaxation time was reduced, to a greater extent in KI cells. EGCg shortened time to peak Ca2+ and Ca2+ transient decay in Fura-2-loaded WT and KI cardiomyocytes. EGCg did not influence phosphorylation of phospholamban. In skinned cardiac muscle strips, EGCg (30 μM) decreased Ca2+ sensitivity in both groups.Conclusion: EGCg hastened relaxation and Ca2+ transient decay to a larger extent in KI than in WT cardiomyocytes. This effect could be partially explained by myofilament Ca2+ desensitization.

The cardiac maladaptive ATF3-dependent cross-talk between cardiomyocytes and macrophages is mediated by the IFNγ-CXCL10-CXCR3 axis

RationalPressure overload induces adaptive and maladaptive cardiac remodeling processes in the heart. Part of the maladaptive process is the cross-talk between cardiomyocytes and macrophages which is dependent on the function of the Activating Transcription Factor 3, ATF3. Yet, the molecular mechanism involved in cardiomyocytes-macrophages communication leading to macrophages recruitment to the heart and cardiac maladaptive remodeling is currently unknown. Methods and resultsIsolated peritoneal macrophages from either wild type or ATF3-KO mice were cultured in serum free medium to collect conditioned medium (CM). CM was used to probe an antibody cytokine/chemokine array. The interferon γ induced protein 10kDa, CXCL10, was found to be enriched in wild type macrophages CM. Wild type cardiomyocytes treated with CXCL10 in vitro, resulted in significant increase in cell volume as compared to ATF3-KO cardiomyocytes. In vivo, pressure overload was induced by phenylephrine (PE) infusion using micro-osmotic pumps. Consistently, CXCL11 (CXCL10 competitive agonist) and CXCL10 receptor antagonist (AMG487) attenuated PE-dependent maladaptive cardiac remodeling. Significantly, we show that the expression of the CXCL10 receptor, CXCR3, is suppressed in cardiomyocytes and macrophages derived from ATF3-KO mice. CXCR3 is positively regulated by ATF3 through an ATF3 transcription response element found in its proximal promoter. Finally, mice lacking CXCR3 display a significant reduction of cardiac remodeling processes following PE infusion. ConclusionsChronic PE infusion results in a unique cardiomyocytes-macrophages cross-talk that is mediated by IFNγ. Subsequently, macrophages that are recruited to the heart secrete CXCL10 resulting in maladaptive cardiac remodeling mediated by the CXCR3 receptor. ATF3-KO mice escape from PE-dependent maladaptive cardiac remodeling by suppressing the IFNγ-CXCL10-CXCR3 axis at multiple levels.

Differential roles of caveolin-1 in ouabain-induced Na+/K+-ATPase cardiac signaling and contractility.

Binding of ouabain to cardiac Na+/K+-ATPase initiates cell signaling and causes contractility in cardiomyocytes. It is widely accepted that caveolins, structural proteins of caveolae, have been implicated in signal transduction. It is known that caveolae play a role in Na+/K+-ATPase functions. Regulation of caveolin-1 in ouabain-mediated cardiac signaling and contractility has never been reported. The aim of this study is to compare ouabain-induced cardiac signaling and contractility in wild-type (WT) and caveolin-1 knockout (cav-1 KO) mice. In contrast with WT cardiomyocytes, ouabain-induced signaling e.g., activation of phosphoinositide 3-kinase-α/Akt and extracellular signal-regulated kinases (ERK)1/2, and hypertrophic growth were significantly reduced in cav-1 KO cardiomyocytes. Interactions of the Na+/K+-ATPase α1-subunit with caveolin-3 and the Na+/K+-ATPase α1-subunit with PI3K-α were also decreased in cav-1 KO cardiomyocytes. The results from cav-1 KO mouse embryonic fibroblasts also proved that cav-1 significantly attenuated ouabain-induced ERK1/2 activation without alteration in protein and cholesterol distribution in caveolae/lipid rafts. Intriguingly, the effect of ouabain induced positive inotropy in vivo (via transient infusion of ouabain, 0.48 nmol/g body wt) was not attenuated in cav-1 KO mice. Furthermore, ouabain (1-100 μM) induced dose-dependent contractility in isolated working hearts from WT and cav-1 KO mice. The effects of ouabain on contractility between WT and cav-1 KO mice were not significantly different. These results demonstrated differential roles of cav-1 in the regulation of ouabain signaling and contractility. Signaling by ouabain, in contrast to contractility, may be a redundant property of Na+/K+-ATPase.

Essential Opposite Roles of ERK and Akt Signaling in Cardiac Steroid-Induced Increase in Heart Contractility.

Interaction of cardiac steroids (CS) with the Na(+), K(+)-ATPase elicits, in addition to inhibition of the enzyme's activity, the activation of intracellular signaling such as extracellular signal-regulated (ERK) and protein kinase B (Akt). We hypothesized that the activities of these pathways are involved in CS-induced increase in heart contractility. This hypothesis was tested using in vivo and ex vivo wild type (WT) and sarcoplasmic reticulum Ca(2+) atpase1a-deficient zebrafish (accordion, acc mutant) experimental model. Heart contractility was measured in vivo and in primary cardiomyocytes in WT zebrafish larvae and acc mutant. Ca(2+) transients were determined ex vivo in adult zebrafish hearts. CS dose dependently augmented the force of contraction of larvae heart muscle and cardiomyocytes and increased Ca(2+) transients in WT but not in acc mutant. CS in vivo increased the phosphorylation rate of ERK and Akt in the adult zebrafish heart of the two strains. Pretreatment of WT zebrafish larvae or cardiomyocytes with specific MAPK inhibitors completely abolished the CS-induced increase in contractility. On the contrary, pretreatment with Akt inhibitor significantly enhanced the CS-induced increase in heart contractility both in vivo and ex vivo without affecting CS-induced Ca(2+) transients. Furthermore, pretreatment of the acc mutant larvae or cardiomyocytes with Akt inhibitor restored the CS-induced increase in heart contractility also without affecting Ca(2+) transients. These results support the notion that the activity of MAPK pathway is obligatory for CS-induced increases in heart muscle contractility. Akt activity, on the other hand, plays a negative role, via Ca(2+) independent mechanisms, in CS action. These findings point to novel potential pharmacological intervention to increase CS efficacy.

Abstract 12746: Impaired Autophagic Flux in Ischemic/Reperfused Diabetic Heart and Its Reversal by a Novel Orally Active Adiponectin Receptor Agonist via AMPK-Mediated Autophagosome Formation and AMPK-Independent Autophagosome Clearance

Autophagy is increasingly recognized as an important regulator of myocardial ischemia/reperfusion (MI/R) injury. However, whether and how autophagy is altered in the ischemic/reperfused diabetic heart remains unknown. Normal control (NC), high-fat-diet-induced diabetic (HFD), and adiponectin (APN) knockout (APNKO) mice were subjected to MI/R (30 minutes/3-24 hours) and treated with vehicle or AdipoRon (50 mg/kg gavage, 10 minutes before MI; or 10 mg/kg IP, 10 min before R). Compared to NC mice, autophagosome formation was significantly inhibited and autophagosome clearance was nearly completely blocked in the HFD heart after MI/R (P<0.01). APNKO largely reproduced the phenotypic alterations observed in the HFD heart, with both autophagosome formation and clearance inhibited after MI/R (P<0.01 vs. WT). Treatment of HFD and APKNO mice with AdipoRon stimulated autophagosome formation, increased autophagosome clearance, reduced infarct size, and improved cardiac function (P<0.01). Mechanistically, AdipoRon caused significant phosphorylation of AMPK, BeclineT119, and VPS34S16, and enhanced expression of lysosome protein LAMP2. Compound C pretreatment abolished AdipoRon-induced BeclineT119 and VPS34S164 phosphorylation without altering AdipoRon-induced LAMP2 expression. In vitro AdipoRon treatment of adult wild type cardiomyocytes enhanced phosphorylation of AMPK, BeclineT119, and VPS34S164. These effects are lost in cardiomyocytes isolated from cardiomyocyte-specific AMPK dominant negative mice (AMPK-DN). In contrast, AdipoRon-stimulated LAMP2 expression was preserved in AMPK-DN cardiomyocytes. Collectively, these results demonstrated for the first time that hypoadiponectinemia during diabetic conditions impairs autophagic flux in cardiomyocytes after MI/R via marked inhibition of both autophagosome clearance and formation. Administration of an orally active APN receptor agonist increased autophagosome formation via AMPK activation, and stimulated autophagosome clearance in an AMPK-independent fashion. Improving autophagic flux by restoring APN signaling systems might be a novel intervention against MI/R injury in diabetic conditions.

Adenine nucleotide translocase 1 overexpression protects cardiomyocytes against hypoxia via increased ERK1/2 and AKT activation

The influence of mitochondrial function on intracellular signalling is currently under intense investigation. In this regard, we analysed the effect of adenine nucleotide translocase 1 (ANT1), which facilitates the exchange of ADP and ATP across the mitochondrial membrane, on cell-protective survival signalling under hypoxia. ANT1 overexpression enhanced the survival rate in hypoxic cardiomyocytes. The effect was related to stabilization of the mitochondrial membrane potential, suppression of caspase 3 activity, and a reduction in DNA fragmentation. Activation of the cell-protective signalling proteins extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (AKT) was substantially higher in hypoxic ANT1-transgenic (ANT1-TG) cardiomyocytes than in wild-type cardiomyocytes. Kinase activation was associated with significantly higher expression of hypoxia-inducible factor 1α, which induces glycolytic pathway to stabilize ATP production. Accordingly, ANT1-TG cardiomyocytes exhibited earlier and stronger activation of lactate dehydrogenase and a higher ATP content. Treatment with PD980559 and triciribine, inhibitors of ERK1/2 and AKT activation, respectively, abolished cell protection in hypoxic ANT1-TG cardiomyocytes. Inhibition of ANT by carboxyatractyloside prevented the increase in ERK1/2 and AKT phosphorylation and eliminated the cell protective program in hypoxic ANT1-TG cardiomyocytes.In conclusion, the cytoprotective effect observed in hypoxic ANT1-overexpressing cardiomyocytes involves an interdependence between ANT1, activation of ERK1/ERK2 and AKT, and induction of the survival processes regulated by these kinases.

Ranolazine antagonizes catecholamine-induced dysfunction in isolated cardiomyocytes, but lacks long-term therapeutic effects in vivo in a mouse model of hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is often accompanied by increased myofilament Ca(2+) sensitivity and diastolic dysfunction. Recent findings indicate increased late Na(+) current density in human HCM cardiomyocytes. Since ranolazine has the potential to decrease myofilament Ca(2+) sensitivity and late Na(+) current, we investigated its effects in an Mybpc3-targeted knock-in (KI) mouse model of HCM. Unloaded sarcomere shortening and Ca(2+) transients were measured in KI and wild-type (WT) cardiomyocytes. Measurements were performed at baseline (1 Hz) and under increased workload (30 nM isoprenaline (ISO), 5 Hz) in the absence or presence of 10 µM ranolazine. KI myocytes showed shorter diastolic sarcomere length at baseline, stronger inotropic response to ISO, and drastic drop of diastolic sarcomere length under increased workload. Ranolazine attenuated ISO responses in WT and KI cells and prevented workload-induced diastolic failure in KI. Late Na(+) current density was diminished and insensitive to ranolazine in KI cardiomyocytes. Ca(2+) sensitivity of skinned KI trabeculae was slightly decreased by ranolazine. Phosphorylation analysis of cAMP-dependent protein kinase A-target proteins and ISO concentration-response measurements on muscle strips indicated antagonism at β-adrenoceptors with 10 µM ranolazine shifting the ISO response by 0.6 log units. Six-month treatment with ranolazine (plasma level >20 µM) demonstrated a β-blocking effect, but did not reverse cardiac hypertrophy or dysfunction in KI mice. Ranolazine improved tolerance to high workload in mouse HCM cardiomyocytes, not by blocking late Na(+) current, but by antagonizing β-adrenergic stimulation and slightly desensitizing myofilaments to Ca(2+). This effect did not translate in therapeutic efficacy in vivo.

Abstract 203: Cardiomyocyte T-tubule Membrane Turns Over, Releasing BIN1 Containing Microparticles into Blood

Bridging integrator 1 (BIN1) is a cardiac muscle protein that folds cardiomyocyte T-tubule membrane. BIN1 is intrinsic to cardiac health, and is reduced in acquired heart failure. Interestingly, we have found that BIN1 is also blood available, and that plasma BIN1 correlates with cardiac function, suggesting cardiac origin of plasma BIN1. We found that low plasma BIN1 correlates with failing muscle and predicts ventricular arrhythmia. However, the paradigm does not exist for an intracellular membrane associate cardiomyocyte protein to be homeostatically turned over into blood. In this study, using a mouse model with cardiac specific deletion of Bin1 gene, we identified with biochemical techniques that plasma BIN1 levels directly correlate with cardiac tissue BIN1 levels, indicating cardiac origin. Furthermore, investigations using both super-resolution fluorescent imaging and flow cytometry analysis revealed that adult ventricular cardiomyocytes constantly release BIN1 into blood via membrane microparticle production. Microparticles are small membrane vesicles shed from plasma membrane of a variety of cell types including platelets, leukocytes, and endothelial cells. Using super-resolution three-dimensional stochastic optical reconstruction microscopy (3D-STORM), we found similar to the blood cells, isolated adult mouse cardiomyocytes release Annexin V positive microparticles with diameters ranging between 0.1 to 1.0 μm. These microparticles also carry BIN1 protein. Flow cytometry was also used to detect and quantify microparticles <1.0 μm in size from medium bathing a pure population of adult mouse cardiomyocytes. BIN1 microparticle release is proportional to actin stability and amount of T-tubule membrane folds. Compared to wild type cardiomyocytes, microparticle release is significantly reduced from myocytes with heterozygous deletion of Bin1 gene. These data indicate that cardiomyocyte membrane undergoes dynamic turnover, releasing T-tubule folds into blood as microparticles. Furthermore, plasma BIN1 can be used as a direct measure of cardiomyocyte health and reserve.

Targeting Mechanically‐Activated NOS Signaling in Dystrophic Cardiomyopathy

Patients deficient in dystrophin, a protein that links the cytoskeleton to the extracellular matrix via the dystrophin‐glycoprotein complex (DGC), exhibit muscular dystrophy, cardiomyopathy, and impaired muscle nitric oxide (NO) production. Our objective was to identify the mechanisms of how the DGC regulates cardiac muscle nitric oxide synthase (NOS) activity. Isolated adult wild‐type (WT) or dystrophin‐deficient mdx mouse cardiomyocytes were plated on deformable membranes, and live‐cell DAF‐FM dye fluorescence and biochemical approaches were used to investigate how the DGC regulates mechanically‐activated NO signaling. 1Hz cyclic stretch of WT cardiomyocytes significantly increased NO production and neuronal NOS (nNOS) S1412 phosphorylation. In contrast, stretch‐induced NO production and nNOS phosphorylation were impaired in mdx cardiomyocytes, suggesting that dystrophin regulates the mechanical activation of NOS. We found that inhibition of AMP‐activated protein kinase (AMPK) inhibited stretch‐induced NO production in WT cardiomyocytes, indicating that AMPK plays a critical role in mechanoactivation of NOS. Importantly, direct pharmacological activation of AMPK increased nNOS S1412 phosphorylation and was sufficient to enhance NO production in mdx cardiomyocytes. We conclude that the DGC regulates the mechanical activation of cardiac NOS, and that pharmacological AMPK activation may be a therapeutic strategy to restore NOS activity in dystrophin‐deficient cardiomyocytes. Supported by the Muscular Dystrophy Association, the University of Michigan CVRE Training Program, and NIH T32 GM‐08322.