Neonatal Mouse Myocytes Research Articles

Mitochondrial calcium is decreased by high glucose and is improved by reduction of protein O‐GlcNAcylation in cardiac myocytes (1078.2)

Increased nuclear protein O‐linked β‐N‐acetylglucosamine glycosylation (O‐GlcNAcylation) mediated by high glucose treatment or the hyperglycemia of diabetes mellitus contributes to cardiac myocyte dysfunction. Mitochondrial proteins in cardiac myocytes are also submitted to O‐GlcNAcylation, however, whether excessive O‐GlcNAcylation alters mitochondrial function is incompletely studied. We have previously demonstrated decreased mitochondrial calcium concentration in cardiac myocytes exposed to high glucose. We hypothesize that excessive O‐GlcNAcylation may be responsible of this maladaptive phenomenon and that reducing excessive O‐GlcNAcylation may return mitochondrial calcium levels to normal. Our objective was to test this hypothesis, therefore, we incubated cultured neonatal mouse myocytes in high glucose (HG, 25mM) for 72 hours and inhibited O‐GlcNAcylation by adenoviral expression of a dominant negative mutant of the O‐GlcNAc transferase (dnOGT). As expected, mitochondrial calcium levels were reduced by 20% after HG exposure but were returned to control values after dnOGT expression. In addition, mitochondrial calcium uptake decreased by 52% in HG and was improved by expression of dnOGT, however, did not reach control values. We examined the mitochondrial calcium uniporter (MCU) and found that it is susceptible to be O‐GlcNAcylated and HG further increased O‐GlcNAcylation. In conclusion, our results suggest that excessive O‐GlcNAcylation in hyperglycemia alters mitochondrial calcium homeostasis by increasing O‐GlcNAcylated MCU.Grant Funding Source: Supported by UC MEXUS and VA‐5 I01BX001121‐02‐

α1A-Adrenergic receptor differentially regulates STAT3 phosphorylation through PKCϵ and PKCδ in myocytes

Previous studies demonstrated α1-adrenergic receptors (ARs) increase STAT3 activation in transfected and non-cardiac primary cell lines. However, the mechanism used by α1-ARs resulting in STAT3 activation is unknown. While other G-protein-coupled receptors (GPCRs) can couple to STAT3, these mechanisms demonstrate coupling through SRC, TYK, Rac, or complex formation with Gq and used only transfected cell lines. Using normal and transgenic mice containing constitutively active mutations (CAM) of the α1A-AR subtype, neonatal mouse myocytes and whole hearts were analyzed for the mechanism to couple to STAT3 activation. α1-ARs stimulated time-dependent increases in p-SRC, p-JAK2, and p-STAT3 in normal neonatal myocytes. Using various kinase inhibitors and siRNA, we determined that the α1A-AR coupled to STAT3 through distinct and unique pathways in neonatal myocytes. We found that PKCϵ inhibition decreased p-ERK and p-Ser STAT3 levels without affecting p-Tyr STAT3. In contrast, we found that PKCδ inhibition affected p-SRC and p-JAK2 resulting in decreased p-Tyr and p-Ser STAT3 levels. We suggest a novel α1A-AR mediated PKCϵ/ERK pathway that regulates the phosphorylation status of STAT3 at Ser-727 while PKCδ couples to SRC/JAK2 to affect Tyr-705 phosphorylation. Furthermore, this pathway has not been previously described in a GPCR system that couples to STAT3. Given cell survival and protective cardiac effects induced by PKC, STAT3 and ERK signaling, our results could explain the neuroprotective and cardiac protective pathways that are enhanced with α1A-AR agonism.

Protein kinase C downregulates IKs by stimulating KCNQ1-KCNE1 potassium channel endocytosis

The slow-activating cardiac repolarization K(+) current (I(Ks)), generated by the KCNQ1-KCNE1 potassium channel complex, is controlled via sympathetic and parasympathetic regulation in vivo. Inherited KCNQ1 and KCNE1 mutations predispose to ventricular fibrillation and sudden death, often triggered by exercise or emotional stress. Protein kinase C (PKC), which is activated by α1 adrenergic receptor stimulation, is known to downregulate I(Ks) via phosphorylation of KCNE1 serine 102, but the underlying mechanism has remained enigmatic. We previously showed that KCNE1 mediates dynamin-dependent endocytosis of KCNQ1-KCNE1 complexes. This study sought to determine the potential role of endocytosis in I(Ks) downregulation by PKC. We utilized patch clamping and fluorescence microscopy to study Chinese hamster ovary (CHO) cells coexpressing KCNQ1, KCNE1, and wild-type or dominant-negative mutant (K44A) dynamin 2, and neonatal mouse ventricular myocytes. The PKC activator phorbol 12-myristate 13-acetate (PMA) decreased I(Ks) density by >60% (P < .05) when coexpressed with wild-type dynamin 2 in CHO cells, but had no effect when coexpressed with K44A-dynamin 2. Thus, functional dynamin was required for downregulation of I(Ks) by PKC activation. PMA increased KCNQ1-KCNE1 endocytosis in CHO cells expressing wild-type dynamin 2, but had no effect on KCNQ1-KCNE1 endocytosis in CHO cells expressing K44A-dynamin 2, determined using the Pearson correlation coefficient to quantify endosomal colocalization of KCNQ1 and KCNE1 with internalized fluorescent transferrin. KCNE1-S102A abolished the effect of PMA on I(Ks) currents and endocytosis. Importantly, PMA similarly stimulated endocytosis of endogenous KCNQ1 and KCNE1 in neonatal mouse myocytes. PKC activation downregulates I(Ks) by stimulating KCNQ1-KCNE1 channel endocytosis.

The human CASQ2 mutation K206N is associated with hyperglycosylation and altered cellular calcium handling

Mutations in the human cardiac calsequestrin gene (CASQ2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT-2). This inherited disorder is characterized by life-threatening arrhythmias induced by physical and emotional stress in young patients. Here we identified a novel heterozygous missense mutation (K206N) in the CASQ2 gene in a symptomatic family in which one member died of cardiac arrest. The functional properties of CSQK206N were investigated in comparison to the wild-type form of CASQ2 (CSQWT) by expression in eukaryotic cell lines and neonatal mouse myocytes. The mutation created an additional N-glycosylation site resulting in a higher molecular weight form of the recombinant protein on immunoblots. The mutation reduced the Ca2+ binding capacity of the protein and exhibited an altered aggregation state. Consistently, CSQK206N-expressing myocytes exhibited an impaired response to caffeine administration, suggesting a lower Ca2+ load of the sarcoplasmic reticulum (SR). The interaction of the mutated CSQ with triadin and the protein levels of the ryanodine receptor were unchanged but the maximal specific [3H]ryanodine binding was increased in CSQK206N-expressing myocytes, suggesting a higher opening state of the SR Ca2+ release channel. Myocytes with expression of CSQK206N showed a higher rate of spontaneous SR Ca2+ releases under basal conditions and after β-adrenergic stimulation. We conclude that CSQK206N caused a reduced Ca2+ binding leading to an abnormal regulation of intracellular Ca2+ in myocytes. This may then contribute to the increased propensity to trigger spontaneous Ca2+ transients in CSQK206N-expressing myocytes.

The calsequestrin mutation CASQ2‐K206N affects sarcoplasmic reticulum Ca handling and causes catecholaminergic polymorphic ventricular tachycardia

Catecholamine‐induced polymorphic ventricular tachycardia (CPVT) is a familial disorder caused by calsequestrin 2 (CASQ2) gene mutations. Here, we report the identification of a new CASQ2 mutation, K206N, which transmitted autosomally and is associated with CPVT and sudden cardiac death in a young patient. To find a causal link between K206N and the CPVT phenotype, we studied biochemical and physiological effects of K206N overexpression, in comparison to the native form (NT) of the calsequestrin protein (CSQ), in both insect cells and isolated neonatal mouse myocytes by use of recombinant baculoviruses and adenoviruses, respectively. The CSQ mutation was associated with the insertion of a second glycosylation site resulting in a higher mobility form compared to NT. Consistently, the mutated CSQ colocalized with mannosidase II, whereas NT did not. Thus, hyperglycosylation of K206N may trigger the lower Ca binding (by 54%) observed in 45Ca overlay blots. Moreover, we found a higher binding (by 40%) of the mutated CSQ to a GST‐triadin fusion protein in pull‐down assays. [3H]ryanodine binding was increased by 115% in homogenates of myocytes infected with K206N compared to NT, suggesting a higher open probability of the SR Ca release channel. In line with these findings, myocytes with K206N overexpression had a higher rate of spontaneous Ca transients, when exposed to isoproterenol.The data of the present study demonstrate that overexpression of K206N in myocytes causes an abnormal Ca handling and therefore, may predispose to the CPVT phenotype observed in the human patient.This study was supported by the Deutsche Forschungsgemeinschaft (Ki 653/13‐1/2).

Molecular and Functional Characterization of Novel Glycerol-3-Phosphate Dehydrogenase 1–Like Gene ( GPD1-L ) Mutations in Sudden Infant Death Syndrome

Autopsy-negative sudden unexplained death, including sudden infant death syndrome, can be caused by cardiac channelopathies such as Brugada syndrome (BrS). Type 1 BrS, caused by mutations in the SCN5A-encoded sodium channel, accounts for approximately 20% of BrS. Recently, a novel mutation in the glycerol-3-phosphate dehydrogenase 1-like gene (GPD1-L) disrupted trafficking of SCN5A in a multigenerational family with BrS. We hypothesized that mutations in GPD1-L may be responsible for some cases of sudden unexplained death/sudden infant death syndrome. Using denaturing high-performance liquid chromatography and direct DNA sequencing, we performed comprehensive open-reading frame/splice site mutational analysis of GPD1-L on genomic DNA extracted from necropsy tissue of 83 unrelated cases of sudden unexplained death (26 females, 57 males; average age, 14.6+/-10.7 years; range, 1 month to 48 years). A putative, sudden unexplained death-associated GPD1-L missense mutation, E83K, was discovered in a 3-month-old white boy. Further mutational analysis was then performed on genomic DNA derived from a population-based cohort of 221 anonymous cases of sudden infant death syndrome (84 females, 137 males; average age, 3+/-2 months; range, 3 days to 12 months), revealing 2 additional mutations, I124V and R273C, in a 5-week-old white girl and a 1-month-old white boy, respectively. All mutations occurred in highly conserved residues and were absent in 600 reference alleles. Compared with wild-type GPD1-L, GPD1-L mutations coexpressed with SCN5A in heterologous HEK cells produced a significantly reduced sodium current (P<0.01). Adenovirus-mediated gene transfer of the E83K-GPD1-L mutation into neonatal mouse myocytes markedly attenuated the sodium current (P<0.01). These decreases in current density are consistent with sodium channel loss-of-function diseases like BrS. The present study is the first to report mutations in GPD1-L as a pathogenic cause for a small subset of sudden infant death syndrome via a secondary loss-of-function mechanism whereby perturbations in GPD1-L precipitate a marked decrease in the peak sodium current and a potentially lethal BrS-like proarrhythmic substrate.

Abstract 2968: Postmortem Genetic Testing of the GPD1-L-Encoded Glycerol-3-Phosphate Dehydrogenase 1-Like Protein in Sudden Infant Death Syndrome

Introduction: Approximately 10 – 15% of sudden infant death syndrome (SIDS) may be caused by cardiac channelopathies including Brugada syndrome (BrS). Type 1 BrS (BrS1), due to mutations in the SCN5A-encoded sodium channel, accounts for approximately 20% of BrS. Recently, a novel mutation in glycerol-3-phosphate dehydrogenase 1-like (GPD1-L) disrupted trafficking of SCN5A in a multi-generational family with BrS. We hypothesized that mutations in GPD1-L may be responsible for some cases of SIDS. Methods: Using DHPLC and direct DNA sequencing, we performed comprehensive open reading frame/splice site mutational analysis of GPD1-L on genomic DNA extracted from necropsy tissue of 228 anonymous cases of SIDS (86 females, 142 males, average age = 3 + 2 months, range 6 hours to 12 months). Results: Three putative, SIDS-associated GPD1-L missense mutations, E83K, I124V, and R273C, were discovered in a three-month-old white male, a five-week-old white female, and a one-month-old white male, respectively. All mutations occurred in highly conserved residues and were absent in 600 reference alleles. Compared to wild type GPD1-L, GPD1-L mutations co-expressed with SCN5A in heterologous HEK cells produced a 60% reduction in peak sodium current density (p&lt;0.004). Adenovirus-mediated gene transfer of the E83K-GPD1-L mutation into neonatal mouse myocytes markedly attenuated the sodium current as well (p&lt;0.01). These decreases in current density are consistent with sodium channel loss-of-function diseases like BrS. Conclusion: This study is the first to report mutations in GPD1-L as a pathogenic cause for a small subset (~1%) of SIDS via a secondary loss-of-function mechanism whereby perturbations in GPD1-L precipitate a marked decrease in the peak sodium current and a potentially lethal, BrS-like pro-arrhythmic substrate.

Kv11.1 (ERG1) K+ Channels Localize in Cholesterol and Sphingolipid Enriched Membranes and Are Modulated by Membrane Cholesterol

The localization of ion channels to specific membrane microdomains can impact the functional properties of channels and their role in cellular physiology. We determined the membrane localization of human Kv11.1 (hERG1) α -subunit protein, which underlies the rapidly activating, delayed rectifier K+ current (IKr) in the heart. Immunocytochemistry and membrane fractionation using discontinuous sucrose density gradients of adult canine ventricular tissue showed that Kv11.1 channel protein localized to both the cell surface and T-tubular sarcolemma. Furthermore, density gradient membrane fractionation using detergent (Triton X-100) and non-detergent (OptiPrep) methods from canine ventricular myocytes or HEK293 cells demonstrated that Kv11.1 protein, along with MiRP1 and Kv7.1 (KCNQ1) proteins, localize in cholesterol and sphingolipid enriched membrane fractions. In HEK293 cells, Kv11.1 channels, but not long QT-associated mutant G601S-Kv11.1 channels, also localized to cholesterol and sphingolipid enriched membrane fractions. Depletion of membrane cholesterol from HEK293 cells expressing Kv11.1 channels using methyl-Mβ -cyclodextrin (Mβ CD) caused a positive shift of the voltage dependence of activation and an acceleration of deactivation kinetics of Kv11.1 current (IKv11.1). Cholesterol loading of HEK293 cells reduced the steep voltage dependence of IKv11.1 activation and accelerated the inactivation kinetics of IKv11.1. Incubation of neonatal mouse myocytes in Mβ CD also accelerated the deactivation kinetics of IKr. We conclude that Kv11.1 protein localizes in cholesterol and sphingolipid enriched membranes and that membrane cholesterol can modulate IKv11.1 and IKr.

Cloning and initial characterization of the human cardiac sodium channel (SCN5A) promoter.

Despite the primacy of the sodium current in cardiac electrophysiology and evidence that decreased sodium current is arrhythmogenic in humans, little is known about transcriptional regulation of the underlying gene, SCN5A. We have cloned a 2.7 kb segment of 5'-flanking region of SCN5A and identified multiple transcription initiation sites by primer extension and RNase protection. Transient transfection assays in neonatal mouse myocytes and in Chinese Hamster Ovary (CHO) cells were employed to identify promoter activities. PCR-single stranded conformational polymorphism (SSCP) analysis was used to screen DNA variants in the promoter region. The fragment includes >2 kb of upstream sequence, the 173-bp non-coding exon 1, and a portion of the 16-kb intron 1; the region is highly GC-rich and TATA-less. Transient transfection assays in neonatal mouse myocytes and in CHO cells identified (1) a core promoter in the -261/+140 segment, (2) regions conferring approximately 3-fold decreases from core promoter activity in the 5' upstream region (-261/-454 and -1020/-2109), and approximately 3-fold increases in intron 1 (+255/+410 and+539/+613), and (3) a very strong negative regulatory region between +613 and +754 in intron 1. A core promoter polymorphism, present in 6/142 (4%) of normal alleles screened, increased reporter activity approximately 50% in myocytes but not in CHO cells. The SCN5A promoter includes multiple positive and negative cis-acting elements extending into intron 1. A common polymorphism in this region modulates channel expression in vitro.

Caveolar Localization Dictates Physiologic Signaling of β2-Adrenoceptors in Neonatal Cardiac Myocytes

There is a growing body of evidence that G protein-coupled receptors function in the context of plasma membrane signaling compartments. These compartments may facilitate interaction between receptors and specific downstream signaling components while restricting access to other signaling molecules. We recently reported that beta(1)- and beta(2)-adrenergic receptors (AR) regulate the intrinsic contraction rate in neonatal mouse myocytes through distinct signaling pathways. By studying neonatal myocytes isolated from beta(1)AR and beta(2)AR knockout mice, we found that stimulation of the beta(1)AR leads to a protein kinase A-dependent increase in the contraction rate. In contrast, stimulation of the beta(2)AR has a biphasic effect on the contraction rate. The biphasic effect includes an initial protein kinase A-independent increase in the contraction rate followed by a sustained decrease in the contraction rate that can be blocked by pertussis toxin. Here we present evidence that caveolar localization is required for physiologic signaling by the beta(2)AR but not the beta(1)AR in neonatal cardiac myocytes. Evidence for beta(2)AR localization to caveolae includes co-localization by confocal imaging, co-immunoprecipitation of the beta(2)AR and caveolin 3, and co-migration of the beta(2)AR with a caveolin-3-enriched membrane fraction. The beta(2)AR-stimulated increase in the myocyte contraction rate is increased by approximately 2-fold and markedly prolonged by filipin, an agent that disrupts lipid rafts such as caveolae and significantly reduces co-immunoprecipitation of beta(2)AR and caveolin 3 and co-migration of beta(2)AR and caveolin-3 enriched membranes. In contrast, filipin has no effect on beta(1)AR signaling. These observations suggest that beta(2)ARs are normally restricted to caveolae in myocyte membranes and that this localization is essential for physiologic signaling of this receptor subtype.

Functional consequences of the arrhythmogenic G306R KvLQT1 K+ channel mutant probed by viral gene transfer in cardiomyocytes.

IKs, the slow component of the delayed rectifier potassium current, figures prominently in the repolarization of heart cells. The K+ channel gene KvLQT1 is mutated in the heritable long QT (LQT) syndrome. Heterologous coexpression of KvLQT1 and the accessory protein minK yields an IKs-like current. Nevertheless, the links between KvLQT1 and cardiac IKs are largely inferential. Since the LQT syndrome mutant KvLQT1-G306R suppresses channel activity when coexpressed with wild-type KvLQT1 in a heterologous system, overexpression of this mutant in cardiomyocytes should reduce or eliminate native IKs if KvLQT1 is indeed the major molecular component of this current. To test this idea, we created the adenovirus AdRMGI-KvLQT1-G306R, which overexpresses KvLQT1-G306R channels. In > 60 % of neonatal mouse myocytes, a sizable IKs could be measured using perforated-patch recordings (8.0 +/- 1.6 pA pF-1, n = 13). IKs was increased by forskolin and blocked by clofilium or indapamide but not by E-4031. While cells infected with a reporter virus expressing only green fluorescent protein (GFP) displayed IKs similar to that in uninfected cells, AdRMGI-KvLQT1-G306R-infected cells showed a significantly reduced IKs (2.4 +/- 1.1 pA pF-1, n = 10, P < 0.01) when measured 60-72 h after infection. Similar results were observed in adult guinea-pig myocytes (5.9 +/- 1.2 pA pF-1, n = 9, for control vs. 0.1 +/- 0.1 pA pF-1, n = 5, for AdRMGI-KvLQT1-G306R-infected cells). We conclude that KvLQT1 is the major molecular component of IKs. Our results further establish a dominant-negative mechanism for the G306R LQT syndrome mutation.

3H]Dofetilide Binding to Cardiac Myocytes: Modulation by Extracellular Potassium

The radioligand [3H]dofetilide binds specifically to the delayed rectifier potassium channel and provides a biochemical approach to study interactions of Class III drugs with this channel. However, previous studies have examined the binding of [3H]dofetilide to cardiac myocytes only at extracellular potassium of 135 mm. Because previous electrophysiological studies have shown that hyperkalemia could alter the pharmacological responses to IKrchannel blockers, the hypothesis tested in this study was that changing ionic conditions would alter characteristics of [3H]dofetilide binding. Results: under physiological conditions (Na+135 mm, K+5 mm), [3H]dofetilide bound to two sites on guinea-pig ventricular myocytes (a high-affinity site, Kd26±8 nm, Bmax81±12 fmol/106cells; and a low-affinity site, Kd1.6±0.8μm , Bmax1003±173 fmol/106cells,n=11). Binding properties were not altered by changes in osmolarity or extracellular sodium. However, when extracellular K+was increased to 20 mm, a single binding site was observed with an affinity Kdof 120±12 nmand a Bmaxof 303±57 fmol/106cells (P<0.05;n=6). To establish whether this effect was mediated at the high-affinity site we assessed the effects of elevated extracellular potassium on a biological model, neonatal mouse myocytes, that expressed solely the high-affinity binding sites. The Kdvalues for binding to fetal mouse cardiac myocytes at an extracellular K+of 5 mmand 20 mmwere also significantly different, 29±10 and 230±46 nm, respectively. In conclusion, [3H]dofetilide binding to its high-affinity site is modulated by extracellular potassium.