Knockout Myocytes Research Articles

Abstract Mo068: The role of FGF13 in modulating gap junction protein Connexin-43

Background: Fibroblast growth factor homologous factors (FHFs) are associated with arrhythmias like Brugada syndrome and atrial fibrillation, but their pathological mechanisms remain poorly understood. FHFs are best characterized as regulators of voltage gated sodium channels (VGSCs), however recent studies suggest broader, non-VGSC-related functions, including regulation of microtubules. Whether and how non-VGSC regulatory functions modulate cardiac rhythm is not known. Hypothesis: FGF13 regulates Cx43 trafficking and expression in ventricular cardiomyocytes via destabilizing microtubules. Aims: Building on an unbiased proteomic screen, we investigated if FGF13, the predominant FHF in rodent heart, affects the expression and trafficking of Connexin-43 (Cx43), the predominant gap junction protein in ventricular myocytes. Methods: To identify additional FGF13-dependent functional roles, we employed proximity labeling proteomics in mouse heart with the TurboID biotin ligase fused to FGF13. We used immunoblots, immunohistochemistry, and immunocytochemistry to further characterize the effects of FGF13 ablation on Cx43 pattern distribution and expression. We also leveraged electrophysiology techniques using gap junction inhibitors to detect any Cx43-dependent functional changes in FGF13-ablated cardiomyocytes. Results: The proteomic data revealed that cell junction proteins associated with the intercalated disc (ID) were among the top FGF13 near neighbors, of which Cx43 was heavily enriched, together with microtubule associated proteins (MAPs) known to be critical for cardiomyocyte function and ion channel trafficking. In ventricular cardiomyocytes from constitutive cardiomyocyte-restricted Fgf13 knockout mice, we found markedly perturbed trafficking of Cx43 and reduced colocalization with the ID marker N-Cadherin. Separately, MAP4 abundance was markedly reduced. The addition of colchicine, a microtubule destabilizing drug, to WT cells altered Cx43 distribution pattern similar to that observed in FGF13-ablated cardiomyocytes. Re-expression of FGF13 restored Cx43 trafficking and ID targeting. Electrophysiology experiments using a Cx43 hemichannel inhibitor, Gap19, showed a perturbation of the action potential duration in knockout cardiomyocytes. Isoproterenol-induced action potential prolongation in knockout myocytes was decreased by the addition of Gap19. Conclusions: FGF13 regulates microtubule-based trafficking and targeting of Cx43.

Effects of PMCA1 knockout on ventricular cardiomyocyte calcium homeostasis.

Plasma membrane calcium ATPase 1 (PMCA1), one of two PMCA isoforms expressed in the heart, is considered a minor Ca2+ regulatory transporter. However, this has been difficult to confirm due to lack of specific pharmacological blockers. To test the contribution of PMCA1 to Ca2+ regulation in ventricular tissue, we created a PMCA1 knockout (KO) model by breeding mice floxed at exon 10 of the PMCA1 gene with mice expressing Cre under the MLC2v promotor. The KO efficiency as determined by qPCR showed 60% reduction in PMCA1 transcript in ventricular cardiomyocytes when compared to PMCA1fx/fx mice (referred to as WT). Echocardiography and electrocardiography showed no significant differences in ejection fraction or heart rate in KO compared to WT. However, in field-stimulated isolated ventricular myocytes loaded with the Ca2+ indicator fura-2 AM, we found significant increases in systolic Ca2+ (KO: 1.9±0.5 vs WT: 1.5±0.3 F340/F380, p<0.0001) or Ca2+ transient amplitude (KO: 1.0±0.5 vs WT: 0.6±0.3 F340/F380, p<0.0001, n=30 from 3 mice). There was no change in diastolic Ca2+. We also found an increase in active sarcomere shortening in KO (7.2±3%) vs WT (5.9±3%, p=0.014, n=30), while the contraction and relaxation kinetics did not differ significantly. KO myocytes were less responsive than WT to the β-adrenergic agonist isoproterenol, with smaller proportional increases in systolic Ca2+, delta Ca2+ and myocyte shortening. In patch clamped myocytes, PMCA1 KO myocytes displayed prolonged action potential duration (APD) (KO: 227±100 vs WT: 72±30 ms, p=0.0002, n=10 from 5 mice) and more frequent early afterdepolarizations (EADs) than WT. 80% of KO cells had EADs, whereas there were none in WT. These results suggest that PMCA1 is critical for maintaining normal calcium homeostasis in ventricular cardiomyocytes and could be explored as a potential therapeutic target for Ca2+ regulation.

Acute Genetic Ablation of Cardiac Sodium/Calcium Exchange in Adult Mice: Implications for Cardiomyocyte Calcium Regulation, Cardioprotection, and Arrhythmia.

BackgroundSodium‐calcium (Ca2+) exchanger isoform 1 (NCX1) is the dominant Ca2+ efflux mechanism in cardiomyocytes and is critical to maintaining Ca2+ homeostasis during excitation‐contraction coupling. NCX1 activity has been implicated in the pathogenesis of cardiovascular diseases, but a lack of specific NCX1 blockers complicates experimental interpretation. Our aim was to develop a tamoxifen‐inducible NCX1 knockout (KO) mouse to investigate compensatory adaptations of acute ablation of NCX1 on excitation‐contraction coupling and intracellular Ca2+ regulation, and to examine whether acute KO of NCX1 confers resistance to triggered arrhythmia and ischemia/reperfusion injury.Methods and ResultsWe used the α‐myosin heavy chain promoter (Myh6)‐MerCreMer promoter to create a tamoxifen‐inducible cardiac‐specific NCX1 KO mouse. Within 1 week of tamoxifen injection, NCX1 protein expression and current were dramatically reduced. Diastolic Ca2+ increased despite adaptive reductions in Ca2+ current and action potential duration and compensatory increases in excitation‐contraction coupling gain, sarcoplasmic reticulum Ca2+ ATPase 2 and plasma membrane Ca2+ ATPase. As these adaptations progressed over 4 weeks, diastolic Ca2+ normalized and SR Ca2+ load increased. Left ventricular function remained normal, but mild fibrosis and hypertrophy developed. Transcriptomics revealed modification of cardiovascular‐related gene networks including cell growth and fibrosis. NCX1 KO reduced spontaneous action potentials triggered by delayed afterdepolarizations and reduced scar size in response to ischemia/reperfusion.ConclusionsTamoxifen‐inducible NCX1 KO mice adapt to acute genetic ablation of NCX1 by reducing Ca2+ influx, increasing alternative Ca2+ efflux pathways, and increasing excitation‐contraction coupling gain to maintain contractility at the cost of mild Ca2+‐activated hypertrophy and fibrosis and decreased survival. Nevertheless, KO myocytes are protected against spontaneous action potentials and ischemia/reperfusion injury.

Acute Genetic Ablation of Cardiac Sodium-Calcium Exchange Suppresses Arrhythmogenic Delayed after Depolarizations

Sodium-calcium exchange (NCX) is the dominant calcium (Ca) efflux mechanism in cardiac myocytes. As an electrogenic transporter, NCX is thought to generate the inward current responsible for Ca-dependent arrhythmogenic delayed afterdepolarizations (DADs). To test this hypothesis, we patch-clamped ventricular myocytes isolated from our novel tamoxifen-inducible cardiac-specific NCX knockout (KO) mouse (αMHC-MerCreMer promotor). Tamoxifen (40mg/kg/day IP) was injected for 5 consecutive days in 10 to 12-week-old mice. 4 weeks after injection, we found a 96% decrease of NCX protein expression and an 87% reduction of caffeine-induced NCX current (p< 0.001), with 25% of cells showing no NCX current whatsoever. Sarcoplasmic reticulum Ca load was similar to control. Transient outward current (ITO) and Kv4.2 protein expression were significantly increased (by 60 and 34% respectively, p < 0.01) and ICa was reduced by 47% (p < 0.001). The action potential duration (APD) was shortened when measured at either 10 % (8.6 ± 0.7 ms in control vs 5.5 ± 0.5 ms in KO, p < 0.05) or 90 % (43.9 ± 4.9 ms in control vs 11.1 ± 0.9 ms in KO, p < 0.001) repolarization. In response to a pro-arrhythmic stimulation protocol, spontaneous APs triggered by DADs occurred in 95% of control cells, but only in 35% of NCX KO myocytes, even though we observed no change in the mean number of DADs per cell. Notably, DAD amplitude was significantly reduced in NCX KO mice: 14% of KO cells exhibited DADs with an amplitude ≥ 3mV versus 78 % in control cells. In the 25% of KO cells with no detectable DADs, there were no spontaneous APs. Most likely these were the cells with no NCX activity. Thus, acute genetic reduction/ablation of NCX suppresses DAD frequency/amplitude and subsequent spontaneous AP generation.

Limited Fatty Acid Oxidation (FAO) in CPT2 Knockout Myocytes Associates with Insulin Resistance and Cell Stress: possible role of acylcarnitine lipotoxicity

Lipotoxicity (the accumulation of fat‐derived metabolites that activate cell stress pathways) has been implicated in muscle insulin resistance. We have proposed that lipotoxicity, and in particular accumulation of long‐chain acylcarnitines (LCACs), also contributes to the episodic myopathy and rhabdomyolysis that is evident in persons with inherited disorders of long‐chain FAO. Many studies in the arena of LCAC bioactivity have employed exogenous application of LCACs to cells; few studies have modified endogenously‐produced LCACs to determine myocyte outcomes. Thus, conclusions about the (patho)physiological relevance of LCAC‐associated lipotoxicity have been difficult to determine. Herein, we used CRISPR‐Cas9 technology to knock out carnitine palmitoyltransferase‐2 (CPT2) in murine C2C12 myotubes: as expected, this maneuver led to almost complete inhibition of FAO, plus significant cellular and media accumulation of palmitoylcarnitine in palmitate‐treated cells. In CPT2 KO myotubes, treatment with 500 μM palmitate led to ~2‐fold higher production of IL‐6 (a myocyte cell stress marker) and a ~1.5 to 2‐fold increased release of adenylate kinase (cell death and permeability marker) at 3 and 6 hr post‐treatment (when compared to palmitate‐treated wildtype control cells; n=4/treatment). Furthermore, CPT2 KO myotubes displayed higher sensitivity to palmitate‐induced insulin resistance: the % inhibition in terms of insulin‐induced p‐Akt abundance was 60%, 80%, and 95% in CPT2 KO cells (at 200, 500, and 750 μM palmitate, respectively), vs. 38%, 53%, and 67% in controls (p&lt;0.01, n=8/treatment). The results indicate that limitation of long‐chain FAO by loss of CPT2 in muscle cells elicits cell stress and insulin resistance, consistent with a working model in which lipotoxicity and accumulation of LCAC trigger these outcomes. Further studies are needed to determine if endogenously‐produced LCACs contribute to the phenotypes observed in CPT2 KO myotubes, or if LCAC‐independent mechanisms are involved. It remains to be established whether these phenomena underlie the cardiac and skeletal muscle myopathies typical of inherited disorders of FAO, or contribute to the insulin resistance accompanying type 2 diabetes.Support or Funding InformationThis work was supported by USDA‐ARS Project 6026‐51000‐010‐05S.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Protein Phosphatase 2A Regulates Cardiac Na+ Channels.

Voltage-gated Na+ channel ( INa) function is critical for normal cardiac excitability. However, the Na+ channel late component ( INa,L) is directly associated with potentially fatal forms of congenital and acquired human arrhythmia. CaMKII (Ca2+/calmodulin-dependent kinase II) enhances INa,L in response to increased adrenergic tone. However, the pathways that negatively regulate the CaMKII/Nav1.5 axis are unknown and essential for the design of new therapies to regulate the pathogenic INa,L. To define phosphatase pathways that regulate INa,L in vivo. A mouse model lacking a key regulatory subunit (B56α) of the PP (protein phosphatase) 2A holoenzyme displayed aberrant action potentials after adrenergic stimulation. Unbiased computational modeling of B56α KO (knockout) mouse myocyte action potentials revealed an unexpected role of PP2A in INa,L regulation that was confirmed by direct INa,L recordings from B56α KO myocytes. Further, B56α KO myocytes display decreased sensitivity to isoproterenol-induced induction of arrhythmogenic INa,L, and reduced CaMKII-dependent phosphorylation of Nav1.5. At the molecular level, PP2A/B56α complex was found to localize and coimmunoprecipitate with the primary cardiac Nav channel, Nav1.5. PP2A regulates Nav1.5 activity in mouse cardiomyocytes. This regulation is critical for pathogenic Nav1.5 late current and requires PP2A-B56α. Our study supports B56α as a novel target for the treatment of arrhythmia.

Trpm2 enhances physiological bioenergetics and protects against pathological oxidative cardiac injury: Role of Pyk2 phosphorylation.

The mechanisms by which Trpm2 channels enhance mitochondrial bioenergetics and protect against oxidative stress-induced cardiac injury remain unclear. Here, the role of proline-rich tyrosine kinase 2 (Pyk2) in Trpm2 signaling is explored. Activation of Trpm2 in adult myocytes with H2 O2 resulted in 10- to 21-fold increases in Pyk2 phosphorylation in wild-type (WT) myocytes which was significantly lower (~40%) in Trpm2 knockout (KO) myocytes. Pyk2 phosphorylation was inhibited (~54%) by the Trpm2 blocker clotrimazole. Buffering Trpm2-mediated Ca2+ increase with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) resulted in significantly reduced pPyk2 in WT but not in KO myocytes, indicating Ca2+ influx through activated Trpm2 channels phosphorylated Pyk2. Part of phosphorylated Pyk2 translocated from cytosol to mitochondria which has been previously shown to augment mitochondrial Ca2+ uptake and enhance adenosine triphosphate generation. Although Trpm2-mediated Ca2+ influx phosphorylated Ca2+ -calmodulin kinase II (CaMKII), the CaMKII inhibitor KN93 did not significantly affect Pyk2 phosphorylation in H2 O2 -treated WT myocytes. After ischemia/reperfusion (I/R), Pyk2 phosphorylation and its downstream prosurvival signaling molecules (pERK1/2 and pAkt) were significantly lower in KO-I/R when compared with WT-I/R hearts. After hypoxia/reoxygenation, mitochondrial membrane potential was lower and superoxide level was higher in KO myocytes, and were restored to WT values by the mitochondria-targeted superoxide scavenger MitoTempo. Our results suggested that Ca2+ influx via tonically activated Trpm2 phosphorylated Pyk2, part of which translocated to mitochondria, resulting in better mitochondrial bioenergetics to maintain cardiac health. After I/R, Pyk2 activated prosurvival signaling molecules and prevented excessive increases in reactive oxygen species, thereby affording protection from I/R injury.

Caveolin-3 KO disrupts t-tubule structure and decreases t-tubular ICa density in mouse ventricular myocytes.

Caveolin-3 (Cav-3) is a protein that has been implicated in t-tubule formation and function in cardiac ventricular myocytes. In cardiac hypertrophy and failure, Cav-3 expression decreases, t-tubule structure is disrupted, and excitation-contraction coupling is impaired. However, the extent to which the decrease in Cav-3 expression underlies these changes is unclear. We therefore investigated the structure and function of myocytes isolated from the hearts of Cav-3 knockout (KO) mice. These mice showed cardiac dilatation and decreased ejection fraction in vivo compared with wild-type control mice. Isolated KO myocytes showed cellular hypertrophy, altered t-tubule structure, and decreased L-type Ca2+ channel current (ICa) density. This decrease in density occurred predominantly in the t-tubules, with no change in total ICa, and was therefore a consequence of the increase in membrane area. Cav-3 KO had no effect on L-type Ca2+ channel expression, and C3SD peptide, which mimics the scaffolding domain of Cav-3, had no effect on ICa in KO myocytes. However, inhibition of PKA using H-89 decreased ICa at the surface and t-tubule membranes in both KO and wild-type myocytes. Cav-3 KO had no significant effect on Na+/Ca2+ exchanger current or Ca2+ release. These data suggest that Cav-3 KO causes cellular hypertrophy, thereby decreasing t-tubular ICa density.NEW & NOTEWORTHY Caveolin-3 (Cav-3) is a protein that inhibits hypertrophic pathways, has been implicated in the formation and function of cardiac t-tubules, and shows decreased expression in heart failure. This study demonstrates that Cav-3 knockout mice show cardiac dysfunction in vivo, while isolated ventricular myocytes show cellular hypertrophy, changes in t-tubule structure, and decreased t-tubular L-type Ca2+ current density, suggesting that decreased Cav-3 expression contributes to these changes in cardiac hypertrophy and failure.

Remodeling of repolarization and arrhythmia susceptibility in a myosin-binding protein C knockout mouse model.

Hypertrophic cardiomyopathy (HCM) is one of the most common genetic cardiac diseases and among the leading causes of sudden cardiac death (SCD) in the young. The cellular mechanisms leading to SCD in HCM are not well known. Prolongation of the action potential (AP) duration (APD) is a common feature predisposing hypertrophied hearts to SCD. Previous studies have explored the roles of inward Na+ and Ca2+ in the development of HCM, but the role of repolarizing K+ currents has not been defined. The objective of this study was to characterize the arrhythmogenic phenotype and cellular electrophysiological properties of mice with HCM, induced by myosin-binding protein C (MyBPC) knockout (KO), and to test the hypothesis that remodeling of repolarizing K+ currents causes APD prolongation in MyBPC KO myocytes. We demonstrated that MyBPC KO mice developed severe hypertrophy and cardiac dysfunction compared with wild-type (WT) control mice. Telemetric electrocardiographic recordings of awake mice revealed prolongation of the corrected QT interval in the KO compared with WT control mice, with overt ventricular arrhythmias. Whole cell current- and voltage-clamp experiments comparing KO with WT mice demonstrated ventricular myocyte hypertrophy, AP prolongation, and decreased repolarizing K+ currents. Quantitative RT-PCR analysis revealed decreased mRNA levels of several key K+ channel subunits. In conclusion, decrease in repolarizing K+ currents in MyBPC KO ventricular myocytes contributes to AP and corrected QT interval prolongation and could account for the arrhythmia susceptibility.NEW & NOTEWORTHY Ventricular myocytes isolated from the myosin-binding protein C knockout hypertrophic cardiomyopathy mouse model demonstrate decreased repolarizing K+ currents and action potential and QT interval prolongation, linking cellular repolarization abnormalities with arrhythmia susceptibility and the risk for sudden cardiac death in hypertrophic cardiomyopathy.

Abstract 13412: Differential Insulin-dependent Signaling Pathways Modulated by Hydrogen Peroxide in Cardiac Myocytes

Hydrogen peroxide (H 2 O 2 ) has long been implicated in insulin signal transduction, yet the mechanisms whereby insulin regulates intracellular H 2 O 2 to modulate physiological responses remain obscure. Here we explore the roles of H 2 O 2 in regulation of insulin pathways in cardiac myocytes. We found that insulin inhibits cardiac myocyte beta adrenergic physiological and biochemical responses. We found that catalase (which degrades H 2 O 2 ) completely blocks the ability of insulin to activate the insulin-responsive kinases Akt/mTOR and also to attenuate beta adrenergic receptor-mediated increases in myocyte contractility. These insulin responses are abrogated in a mouse model of type 2 diabetes, establishing a key role for H 2 O 2 -dependent pathways in the diabetic heart. To directly probe intracellular H 2 O 2 responses to insulin in cardiac myocytes, we constructed a novel recombinant AAV9 vector expressing the fluorescent H 2 O 2 biosensor HyPer, which we used to infect mice in vivo . We isolated cardiac myocytes from mice infected with this biosensor and performed quantitative imaging studies. This approach revealed robust insulin-dependent increases in cardiac myocyte H 2 O 2 in response to insulin in wild-type mice. Attenuated H 2 O 2 responses were observed in myocytes isolated from mouse lines deficient in the NADPH oxidase isoforms Nox2 or Nox4 that had been infected with HyPer-AAV9. In Nox2 knockout myocytes, we found that insulin-promoted phosphorylation of kinases Akt and mTor is markedly attenuated, while insulin-dependent inhibition of beta adrenergic contractility is unaffected. In contrast, in cardiac myocytes isolated from the Nox4 knockout mouse, insulin-dependent Akt/mTor phosphorylation responses are unaffected, while the ability of insulin to attenuate the beta adrenergic increase in myocyte contractility was lost. These studies represent an important advance in our understanding of insulin action in the heart by identifying a proximal point of bifurcation in insulin signaling via the differential activation of two NADPH oxidase isoforms. The work also provides insight into mechanisms of diabetic cardiomyopathy, and suggest that insulin resistance in the diabetic heart may unmask potentially deleterious beta adrenergic responses.

Abstract 122: Pkialpha Protects Hearts From Beta-adrenergic Overstimulation

Introduction: Protein kinase inhibitor peptide α (PKIα) isoforms are abundantly expressed endogenous inhibitors of PKA in the heart. The physiological and pathophysiological relevance of PKIα has not been studied. Hypothesis: PKIα inhibits PKA activity in normal and stressed (post-myocardial infarction (MI)) hearts. Methods: PKIα knockout (KO) and c57bl/6 control (CTR) mice were used to test if PKIα affected cardiac β-adrenergic response and if played a role in post-MI hearts. Results: Protein abundance of PKIα was significantly increased in failing human and post-MI mouse ventricular tissue. The knockout of PKIα in mice did not affect the expression of the other two PKI isoforms. Nor did it change the expression of PKA regulatory and catalytic subunits. PKIα KO mice did not show alterations in basal cardiac morphology, function and life span of mice. Intra-LV hemodynamic measurements in response to different doses of isoproterenol showed that the loss of PKIα sensitizes the heart in response to isoproterenol (ISO) with a lower EC50 and the maximum response was increased more in KO mice than in control mice. ISO (10nM) increased myocyte contraction and Ca2+ transient amplitudes more in KO myocytes than control myocytes though 100nM ISO increased myocyte contraction and Ca2+ transients amplitude to the same. At the ISO concentration of 10nM, ISO increased more phosphorylation of the Ser16 site (PKA site) on phospholamban. In post-MI animals, the loss of PKIα enhances cardiac function at 1 week after MI but caused more myocardial infarction and functional depression at 8 weeks after MI. Conclusions: PKIα is an important regulator of cardiac β-adrenergic responses by antagonizing PKA. This mechanism could be protective to avoid β-adrenergic overstimulation, especially in post-MI patients and animal models.

Nitric oxide mediates stretch-induced Ca2+ oscillation in smooth muscle

The stretching of smooth muscle tissue modulates contraction through augmentation of Ca(2+) transients, but the mechanism underlying stretch-induced Ca(2+) transients is still unknown. We found that mechanical stretching and maintenance of mouse urinary bladder smooth muscle strips and single myocytes at 30% and 18% beyond the initial length, respectively, resulted in Ca(2+) oscillations. Experiments indicated that mechanical stretching remarkably increased the production of nitric oxide (NO) as well as the amplitude and duration of muscle contraction. Stretch-induced Ca(2+) oscillations and contractility increases were completely abolished by the NO inhibitor L-NAME or eNOS (also known as NOS3) gene inactivation. Moreover, exposure of eNOS-knockout myocytes to exogenous NO donor induced Ca(2+) oscillations. The stretch-induced Ca(2+) oscillations were greatly inhibited by the selective inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor xestospongin C and partially inhibited by ryanodine. Moreover, the stretch-induced Ca(2+) oscillations were also suppressed by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, but not by the soluble guanylyl cyclase (sGC) inhibitor ODQ. These results suggest that stretching myocyte and maintenance at a certain length results in Ca(2+) oscillations that are NO dependent , and sGC and cGMP independent, and results from the activation of PI3K in smooth muscle.

Regulation of L-type calcium channel by phospholemman in cardiac myocytes

We evaluated whether phospholemman (PLM) regulates L-type Ca2+ current (ICa) in mouse ventricular myocytes. Expression of α1-subunit of L-type Ca2+ channels between wild-type (WT) and PLM knockout (KO) hearts was similar. Compared to WT myocytes, peak ICa (at −10mV) from KO myocytes was ~41% larger, the inactivation time constant (τinact) of ICa was ~39% longer, but deactivation time constant (τdeact) was similar. In the presence of isoproterenol (1μM), peak ICa was ~48% larger and τinact was ~144% higher in KO myocytes. With Ba2+ as the permeant ion, PLM enhanced voltage-dependent inactivation but had no effect on τdeact. To dissect the molecular determinants by which PLM regulated ICa, we expressed PLM mutants by adenovirus-mediated gene transfer in cultured KO myocytes. After 24h in culture, KO myocytes expressing green fluorescent protein (GFP) had significantly larger peak ICa and longer τinact than KO myocytes expressing WT PLM; thereby independently confirming the observations in freshly isolated myocytes. Compared to KO myocytes expressing GFP, KO myocytes expressing the cytoplasmic domain truncation mutant (TM43), the non-phosphorylatable S68A mutant, the phosphomimetic S68E mutant, and the signature PFXYD to alanine (ALL5) mutant all resulted in lower peak ICa. Expressing PLM mutants did not alter expression of α1-subunit of L-type Ca2+ channels in cultured KO myocytes. Our results suggested that both the extracellular PFXYD motif and the transmembrane domain of PLM but not the cytoplasmic tail were necessary for regulation of peak ICa amplitude. We conclude that PLM limits Ca2+ influx in cardiac myocytes by reducing maximal ICa and accelerating voltage-dependent inactivation.

GW24-e0050 Stretch current-induced abnormal impulses in CaMKII & knockout mouse ventricular myocytes

ObjectivesCaMKII activation is pro-arrhythmic in heart failure where myocardium is stretched. However, the arrhythmogenic role of CaMKII in stretched ventricle has not been well understood. We tested abnormal impulse inducibility...

While systolic cardiomyocyte function is preserved, diastolic myocyte function and recovery from acidosis are impaired in CaMKIIδ-KO mice

ObjectiveCaMKII contributes to impaired contractility in heart failure by inducing SR Ca2+-leak. CaMKII-inhibition in the heart was suggested to be a novel therapeutic principle. Different CaMKII isoforms exist. Specifically targeting CaMKIIδ, the dominant isoform in the heart, could be of therapeutic potential without impairing other CaMKII isoforms. RationaleWe investigated whether cardiomyocyte function is affected by isoform-specific knockout (KO) of CaMKIIδ under basal conditions and upon stress, i.e. upon ß-adrenergic stimulation and during acidosis. ResultsSystolic cardiac function was largely preserved in the KO in vivo (echocardiography) corresponding to unchanged Ca2+-transient amplitudes and isolated myocyte contractility in vitro. CaMKII activity was dramatically reduced while phosphatase-1 inhibitor-1 was significantly increased. Surprisingly, while diastolic Ca2+-elimination was slower in KO most likely due to decreased phospholamban Thr-17 phosphorylation, frequency-dependent acceleration of relaxation was still present. Despite decreased SR Ca2+-reuptake at lower frequencies, SR Ca2+-content was not diminished, which might be due to reduced diastolic SR Ca2+-loss in the KO as a consequence of lower RyR Ser-2815 phosphorylation. Challenging KO myocytes with isoproterenol showed intact inotropic and lusitropic responses. During acidosis, SR Ca2+-reuptake and SR Ca2+-loading were significantly impaired in KO, resulting in an inability to maintain systolic Ca2+-transients during acidosis and impaired recovery. ConclusionsInhibition of CaMKIIδ appears to be safe under basal physiologic conditions. Specific conditions exist (e.g. during acidosis) under which CaMKII-inhibition might not be helpful or even detrimental. These conditions will have to be more clearly defined before CaMKII inhibition is used therapeutically.

The second member of transient receptor potential-melastatin channel family protects hearts from ischemia-reperfusion injury

The second member of the transient receptor potential-melastatin channel family (TRPM2) is expressed in the heart and vasculature. TRPM2 channels were expressed in the sarcolemma and transverse tubules of adult left ventricular (LV) myocytes. Cardiac TRPM2 channels were functional since activation with H2O2 resulted in Ca(2+) influx that was dependent on extracellular Ca(2+), was significantly higher in wild-type (WT) myocytes compared with TRPM2 knockout (KO) myocytes, and inhibited by clotrimazole in WT myocytes. At rest, there were no differences in LV mass, heart rate, fractional shortening, and +dP/dt between WT and KO hearts. At 2-3 days after ischemia-reperfusion (I/R), despite similar areas at risk and infarct sizes, KO hearts had lower fractional shortening and +dP/dt compared with WT hearts. Compared with WT I/R myocytes, expression of the Na(+)/Ca(2+) exchanger (NCX1) and NCX1 current were increased, expression of the α1-subunit of Na(+)-K(+)-ATPase and Na(+) pump current were decreased, and action potential duration was prolonged in KO I/R myocytes. Post-I/R, intracellular Ca(2+) concentration transients and contraction amplitudes were equally depressed in WT and KO myocytes. After 2 h of hypoxia followed by 30 min of reoxygenation, levels of ROS were significantly higher in KO compared with WT LV myocytes. Compared with WT I/R hearts, oxygen radical scavenging enzymes (SODs) and their upstream regulators (forkhead box transcription factors and hypoxia-inducible factor) were lower, whereas NADPH oxidase was higher, in KO I/R hearts. We conclude that TRPM2 channels protected hearts from I/R injury by decreasing generation and enhancing scavenging of ROS, thereby reducing I/R-induced oxidative stress.

Investigating the Effects of a Cardiotoxic Drug on Calcium Homeostasis in the Heart

Contractile dysfunction and arrhythmogenic activity can arise if there are perturbations to calcium (Ca) homeostasis in the heart, such as in response to cardiotoxic substances. This investigation focuses on changes in cellular Ca homeostasis in response to the acute application of the cardiotoxic drug Clozapine (an atypical anti-psychotic).Rat ventricular myocytes were isolated and stimulated under voltage clamp control. Various parameters of Ca handling were measured both in control conditions and in the presence of 10 μM clozapine. Ca-sensitive indicators fluo3-AM or fura-2 were used to quantify cytosolic Ca. Sarcoplasmic reticulum (SR) Ca content was measured by applying 10 mM caffeine. Activity of Ca extrusion mechanisms (SERCA - sarco-endoplasmic reticulum ATP-ase, NCX - sodium-calcium exchanger) were calculated from Ca transient decay rates. The current-voltage (I-V) relationship of the L-type Ca channel was also measured.Acute application of clozapine significantly reduced mean Ca transient amplitude by 48% and L-type Ca current (ICaL) density by 50%. SR content was decreased by 18 % and SERCA activity (rate constant) was reduced by 34 %. NCX rate constant was reduced by 17%. The effect of clozapine on SERCA was also present in phospholamban knockout myocytes suggesting that the effect on SERCA is not dependent on phospholamban.It remains unclear if or how the observed effects on Ca handling contribute to the cardiotoxicity of clozapine observed in the clinical setting (arrhythmogenesis and myocarditis). We speculate that effects on ICaL may predispose to refractory heterogeneity and reduced SERCA activity may increase the chance for Ca wave propagation. Current work focuses on effects of clozapine on calcium spark activity and SR threshold for Ca waves.

Stretch Current‐Induced Abnormal Impulses in CaMKIIδ Knockout Mouse Ventricular Myocytes

CaMKII activation is proarrhythmic in heart failure where myocardium is stretched. However, the arrhythmogenic role of CaMKII in stretched ventricle has not been well understood. We tested abnormal impulse inducibility by stretch current in myocytes isolated from CaMKIIδ knockout (KO) mouse left ventricle (LV) where CaMKII activity is reduced by ≈ 62%. Action potentials were recorded by whole-cell patch clamp, and abnormal impulses were induced in LV myocytes by a simulation of stretch-activated channel (SAC) current. SAC activation failed to induce abnormal impulses in wild type (WT) myocytes but steadily produced early after-depolarizations and automaticity in KO myocytes in which an increase in L-type calcium channel (LTCC) current (I(Ca)) and a reduction of sarcoplasmic reticulum Ca(2+) leak and action potential duration (APD) were observed. The abnormal impulses were not suppressed by CaMKII inhibitor AIP whereas a low concentration of nifedipine eliminated abnormal impulses without shortening APD, implicating I(Ca) in promoting stretch-induced abnormal impulses. In addition, APD prolongation by LTCC opener S(-)Bay K 8644 or isoproterenol facilitated abnormal impulse induction in WT ventricular myocytes even in the presence of CaMKII inhibitor AIP, whereas APD prolongation by K(+) channel blocker 4-aminopyridine promoted abnormal impulses in KO myocytes but not in WT myocytes. I(Ca) activation plays a central role in stretch-induced abnormal impulses and APD prolongation is arrhythmogenic only when I(Ca) is highly activated. At increased I(Ca) activation, CaMKII inhibition cannot suppress abnormal impulse induction.

Different roles of the cardiac Na+/Ca2+-exchanger in ouabain-induced inotropy, cell signaling, and hypertrophy

Previous studies have shown that digitalis drugs, acting as specific inhibitors of cardiac Na(+)/K(+)-ATPase, not only cause positive inotropic effects, but also activate cell signaling pathways that lead to cardiac myocyte hypertrophy. A major aim of this work was to assess the role of Na(+)/Ca(2+)-exchanger, NCX1, in the above two seemingly related drug effects. Using a mouse with ventricular-specific knockout (KO) of NCX1, ouabain-induced positive inotropy that was evident in isolated wild-type (Wt) hearts was clearly reduced in KO hearts. Ouabain also increased Ca(2+) transient amplitudes in Wt myocytes, but not in KO myocytes. Ouabain-induced activations of ERK 1/2 were noted in Wt myocytes, but not in KO myocytes; however, ouabain activated PI3K1A and Akt in both Wt and KO myocytes. Protein synthesis rate, as a measure of hypertrophy, was increased by ouabain in Wt and KO myocytes; these drug effects were prevented by a PI3K inhibitor but not by a MEK/ERK inhibitor. Hypertrophy caused by ET-1, but not that induced by ouabain, was accompanied by upregulation of BNP gene in Wt and KO myocytes. The findings indicate 1) the necessity of NCX1 for positive inotropic action of ouabain; 2) the irrelevance of NCX1 and ERK 1/2 activation to ouabain-induced hypertrophy; and 3) that hypertrophy caused by ouabain through the activation of PI3K1A/Akt pathway is likely to be beneficial to the heart.

Abstract 19461: Endogenous Protein Kinase Inhibitor Peptide (pki) Regulates β-Adrenergic Responses of the Heart

Introduction: Protein kinase inhibitor peptide (PKI) isoforms are endogenous inhibitors of PKA and existing in the heart. The physiological relevance of PKI has not been studied. Aims: We will determine the role of PKIα (one of the major PKI isoforms in the heart) in β-adrenergic regulation of the heart with PKIα knockout (KO) versus c57bl/6 control (CTR) mice. Methods: In vivo cardiac function was evaluated by conventional echocardiography (ECHO) in PKIα KO mice. In vivo cardiac β-adrenergic response was determined by intra-LV hemodynamic measurements in response to different doses of isoproterenol (10-16g/g BW∼10-5g/g BW). Isolated ventricular myocytes were used to determine cellular responses to β-adrenergic agonist, isoproterenol (ISO, 1nM, 10nM and 100nM). Western blotting was used to determine phosphorylation levels of PKA-targets in the heart. Results: (1) The loss of PKI did not change basal cardiac morphology, function and life span of mice; (2) ISO (2μ g/g BW) enhanced cardiac function more in KO mice than in CTR mice when evaluated by ECHO and intra-LV hemodynamic measurements; (3) The loss of PKIα sensitizes the heart in response to ISO (Log[EC50] shifted from -9.2 to -10.8) and the maximum dp/dt increases more (by 25%) in KO mice than in CTR mcie; (4) PKIa KO myocytes were more sensitive to ISO in that 10nM ISO increased myocyte contraction and Ca2+ transient amplitudes more in KO myocytes; (5). At the ISO concentration of 10-9M, ISO increased more phosphorylation of the Ser16 site (PKA site) on phospholamban. Conclusion: PKI plays an important role in regulating cardiac β-adrenergic responses by antagonizing PKA.