Murine Ventricular Myocytes Research Articles

RGS7 balances acetylation/de-acetylation of p65 to control chemotherapy-dependent cardiac inflammation.

Cardiotoxicity remains a major limitation in the clinical utility of anthracycline chemotherapeutics. Regulator of G-protein Signaling 7 (RGS7) and inflammatory markers are up-regulated in the hearts of patients with a history of chemotherapy particularly those with reduced left-ventricular function. RGS7 knockdown in either the murine myocardium or isolated murine ventricular cardiac myocytes (VCM) or cultured human VCM provided marked protection against doxorubicin-dependent oxidative stress, NF-κB activation, inflammatory cytokine production, and cell death. In exploring possible mechanisms causally linking RGS7 to pro-inflammatory signaling cascades, we found that RGS7 forms a complex with acetylase Tip60 and deacetylase sirtuin 1 (SIRT1) and controls the acetylation status of the p65 subunit of NF-κB. In VCM, the detrimental impact of RGS7 could be mitigated by inhibiting Tip60 or activating SIRT1, indicating that the ability of RGS7 to modulate cellular acetylation capacity is critical for its pro-inflammatory actions. Further, RGS7-driven, Tip60/SIRT1-dependent cytokines released from ventricular cardiac myocytes and transplanted onto cardiac fibroblasts increased oxidative stress, markers of transdifferentiation, and activity of extracellular matrix remodelers emphasizing the importance of the RGS7-Tip60-SIRT1 complex in paracrine signaling in the myocardium. Importantly, while RGS7 overexpression in heart resulted in sterile inflammation, fibrotic remodeling, and compromised left-ventricular function, activation of SIRT1 counteracted the detrimental impact of RGS7 in heart confirming that RGS7 increases acetylation of SIRT1 substrates and thereby drives cardiac dysfunction. Together, our data identify RGS7 as an amplifier of inflammatory signaling in heart and possible therapeutic target in chemotherapeutic drug-induced cardiotoxicity.

Transmembrane protein 117 knockdown protects against angiotensin-II-induced cardiac hypertrophy

Mitochondrial dysfunction plays a critical role in the pathogenesis of pathological cardiac hypertrophy. Transmembrane protein 117 modulate mitochondrial membrane potential that may be involved in the regulation of oxidative stress and mitochondrial function. However, its role in the development of angiotensin II (Ang-II)-induced cardiac hypertrophy is unclear. Cardiac-specific TMEM117-knockout and control mice were subjected to cardiac hypertrophy induced by Ang-II infusion. Small-interfering RNAs against TMEM117 or adenovirus-based plasmids encoding TMEM117 were delivered into left ventricles of mice or incubated with neonatal murine ventricular myocytes (NMVMs) before Ang-II stimulation. We found that TMEM117 was upregulated in hypertrophic hearts and cardiomyocytes and TMEM117 deficiency attenuated Ang-II-induced cardiac hypertrophy in vivo. Consistently, the in vitro data demonstrated that Ang-II-induced cardiomyocyte hypertrophy significantly alleviated by TMEM117 knockdown. Conversely, overexpression of TMEM117 exacerbated cardiac hypertrophy and dysfunction. An Ang II-induced increase in cardiac (cardiomyocyte) oxidative stress was alleviated by cardiac-specific knockout (knockdown) of TMEM117 and was worsened by TMEM117 supplementation (overexpression). In addition, TMEM117 knockout decreased endoplasmic reticulum stress induced by Ang-II, which was reversed by TMEM117 supplementation. Furthermore, TMEM117 deficiency mitigated mitochondrial injury in hypertrophic hearts and cardiomyocyte, which was abolished by TMEM117 supplementation (overexpression). Taken together, these findings suggest that upregulation of TMEM117 contributes to the development of cardiac hypertrophy and the downregulation of TMEM117 may be a new therapeutic strategy for the prevention and treatment of cardiac hypertrophy.

HDAC6 modulates myofibril stiffness and diastolic function of the heart.

Passive stiffness of the heart is determined largely by extracellular matrix and titin, which functions as a molecular spring within sarcomeres. Titin stiffening is associated with the development of diastolic dysfunction (DD), while augmented titin compliance appears to impair systolic performance in dilated cardiomyopathy. We found that myofibril stiffness was elevated in mice lacking histone deacetylase 6 (HDAC6). Cultured adult murine ventricular myocytes treated with a selective HDAC6 inhibitor also exhibited increased myofibril stiffness. Conversely, HDAC6 overexpression in cardiomyocytes led to decreased myofibril stiffness, as did ex vivo treatment of mouse, rat, and human myofibrils with recombinant HDAC6. Modulation of myofibril stiffness by HDAC6 was dependent on 282 amino acids encompassing a portion of the PEVK element of titin. HDAC6 colocalized with Z-disks, and proteomics analysis suggested that HDAC6 functions as a sarcomeric protein deacetylase. Finally, increased myofibril stiffness in HDAC6-deficient mice was associated with exacerbated DD in response to hypertension or aging. These findings define a role for a deacetylase in the control of myofibril function and myocardial passive stiffness, suggest that reversible acetylation alters titin compliance, and reveal the potential of targeting HDAC6 to manipulate the elastic properties of the heart to treat cardiac diseases.

Acute Detubulation of Ventricular Myocytes Amplifies the Inhibitory Effect of Cholinergic Agonist on Intracellular Ca2+ Transients.

Muscarinic receptors expressed in cardiac myocytes play a critical role in the regulation of heart function by the parasympathetic nervous system. How the structural organization of cardiac myocytes affects the regulation of Ca2+ handling by muscarinic receptors is not well-defined. Using confocal Ca2+ imaging, patch-clamp techniques, and immunocytochemistry, the relationship between t-tubule density and cholinergic regulation of intracellular Ca2+ in normal murine ventricular myocytes and myocytes with acute disruption of the t-tubule system caused by formamide treatment was studied. The inhibitory effect of muscarinic receptor agonist carbachol (CCh, 10 μM) on the amplitude of Ca2+ transients, evoked by field-stimulation in the presence of 100 nM isoproterenol (Iso), a β-adrenergic agonist, was directly proportional to the level of myocyte detubulation. The timing of the maximal rate of fluorescence increase of fluo-4, a Ca2+-sensitive dye, was used to classify image pixels into the regions functionally coupled or uncoupled to the sarcolemmal Ca2+ influx (ICa). CCh decreased the fraction of coupled regions and suppressed Ca2+ propagation from sarcolemma inside the cell. Formamide treatment reduced ICa density and decreased sarcoplasmic reticulum (SR) Ca2+ content. CCh did not change SR Ca2+ content in Iso-stimulated control and formamide-treated myocytes. CCh inhibited peak ICa recorded in the presence of Iso by ∼20% in both the control and detubulated myocytes. Reducing ICa amplitude up to 40% by changing the voltage step levels from 0 to –25 mV decreased Ca2+ transients in formamide-treated but not in control myocytes in the presence of Iso. CCh inhibited CaMKII activity, whereas CaMKII inhibition with KN93 mimicked the effect of CCh on Ca2+ transients in formamide-treated myocytes. It was concluded that the downregulation of t-tubules coupled with the diminished efficiency of excitation–contraction coupling, increases the sensitivity of Ca2+ release and propagation to muscarinic receptor-mediated inhibition of both ICa and CaMKII activity.

The Role of T-Tubule Organization in Regulation of Intracellular Ca2+ Handling by Muscarinic Receptor Stimulation in Ventricular Myocytes

Muscarinic receptors expressed in cardiac myocytes play critical role in mediating regulation of heart function by the parasympathetic nervous system. How structural organization of cardiac myocytes affects muscarinic receptor mediated regulation of Ca2+ handling is not well defined. Using confocal Ca2+ imaging and patch-clamp techniques we studied the relationships between t-tubule density and cholinergic regulation of intracellular Ca2+ handling in normal murine ventricular myocytes and in myocytes in which the t-tubule system was disrupted by formamide treatment. Muscarinic receptor agonist CCh (10 μM) significantly inhibited amplitude of Ca2+ transients evoked by electrical field-stimulation in the presence of 100 nM isoproterenol (Iso), a beta-adrenergic agonist, in formamide-treated but not in control ventricular myocytes. Following Iso stimulation, formamide-treated cells exhibited reduced density of the inward Ca2+ current (ICa), decreased sarcoplasmic reticulum (SR) Ca2+ content, and enhanced fractional SR Ca2+ release when compared to control myocytes. CCh did not change SR Ca2+ content in Iso-stimulated formamide-treated myocytes but significantly reduced fractional release. This effect was associated with reduced Ca2+ release at coupled SR Ca2+ release sites and decreased Ca2+ sensitivity of the uncoupled release sites. CCh inhibited peak ICa recorded in a presence of Iso by ∼20 % in both control and detubulated ventricular myocytes. Reducing ICa amplitude by up to 40% by changing voltage step levels from 0 to −25 mV did not affect Ca2+ transients in control myocytes in the presence of Iso. In contrast, in formamide-treated myocytes similar decrease in ICa significantly diminished Ca2+ transients. We conclude that downregulation of the t-tubule system, as often observed in failing hearts, augments the inhibitory effects of muscarinic receptor agonists on Ca2+ transients by reducing functional reserve of excitation-contraction coupling.

Two-hit mechanism of cardiac arrhythmias in diabetic hyperglycaemia: reduced repolarization reserve, neurohormonal stimulation, and heart failure exacerbate susceptibility.

Diabetic hyperglycaemia is associated with increased arrhythmia risk. We aimed to investigate whether hyperglycaemia alone can be accountable for arrhythmias or whether it requires the presence of additional pathological factors. Action potentials (APs) and arrhythmogenic spontaneous diastolic activities were measured in isolated murine ventricular, rabbit atrial, and ventricular myocytes acutely exposed to high glucose. Acute hyperglycaemia increased the short-term variability (STV) of action potential duration (APD), enhanced delayed afterdepolarizations, and the inducibility of APD alternans during tachypacing in both murine and rabbit atrial and ventricular myocytes. Hyperglycaemia also prolonged APD in mice and rabbit atrial cells but not in rabbit ventricular myocytes. However, rabbit ventricular APD was more strongly depressed by block of late Na+ current (INaL) during hyperglycaemia, consistent with elevated INaL in hyperglycaemia. All the above proarrhythmic glucose effects were Ca2+-dependent and abolished by CaMKII inhibition. Importantly, when the repolarization reserve was reduced by pharmacological inhibition of K+ channels (either Ito, IKr, IKs, or IK1) or hypokalaemia, acute hyperglycaemia further prolonged APD and further increased STV and alternans in rabbit ventricular myocytes. Likewise, when rabbit ventricular myocytes were pretreated with isoproterenol or angiotensin II, hyperglycaemia significantly prolonged APD, increased STV and promoted alternans. Moreover, acute hyperglycaemia markedly prolonged APD and further enhanced STV in failing rabbit ventricular myocytes. We conclude that even though hyperglycaemia alone can enhance cellular proarrhythmic mechanisms, a second hit which reduces the repolarization reserve or stimulates G protein-coupled receptor signalling greatly exacerbates cardiac arrhythmogenesis in diabetic hyperglycaemia.

Melatonin alleviates angiotensin-II-induced cardiac hypertrophy via activating MICU1 pathway.

Mitochondrial calcium uptake 1 (MICU1) is a pivotal molecule in maintaining mitochondrial homeostasis under stress conditions. However, it is unclear whether MICU1 attenuates mitochondrial stress in angiotensin II (Ang-II)-induced cardiac hypertrophy or if it has a role in the function of melatonin. Here, small-interfering RNAs against MICU1 or adenovirus-based plasmids encoding MICU1 were delivered into left ventricles of mice or incubated with neonatal murine ventricular myocytes (NMVMs) for 48 h. MICU1 expression was depressed in hypertrophic myocardia and MICU1 knockdown aggravated Ang-II-induced cardiac hypertrophy in vivo and in vitro. In contrast, MICU1 upregulation decreased cardiomyocyte susceptibility to hypertrophic stress. Ang-II administration, particularly in NMVMs with MICU1 knockdown, led to significantly increased reactive oxygen species (ROS) overload, altered mitochondrial morphology, and suppressed mitochondrial function, all of which were reversed by MICU1 supplementation. Moreover, peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α)/MICU1 expression in hypertrophic myocardia increased with melatonin. Melatonin ameliorated excessive ROS generation, promoted mitochondrial function, and attenuated cardiac hypertrophy in control but not MICU1 knockdown NMVMs or mice. Collectively, our results demonstrate that MICU1 attenuates Ang-II-induced cardiac hypertrophy by inhibiting mitochondria-derived oxidative stress. MICU1 activation may be the mechanism underlying melatonin-induced protection against myocardial hypertrophy.

Muscarinic-dependent phosphorylation of the cardiac ryanodine receptor by protein kinase G is mediated by PI3K–AKT–nNOS signaling

Post-translational modifications of proteins involved in calcium handling in myocytes, such as the cardiac ryanodine receptor (RyR2), critically regulate cardiac contractility. Recent studies have suggested that phosphorylation of RyR2 by protein kinase G (PKG) might contribute to the cardioprotective effects of cholinergic stimulation. However, the specific mechanisms underlying these effects remain unclear. Here, using murine ventricular myocytes, immunoblotting, proximity ligation as-says, and nitric oxide imaging, we report that phosphorylation of Ser-2808 in RyR2 induced by the muscarinic receptor agonist carbachol is mediated by a signaling axis comprising phosphoinositide 3-phosphate kinase, Akt Ser/Thr kinase, nitric oxide synthase 1, nitric oxide, soluble guanylate cyclase, cyclic GMP (cGMP), and PKG. We found that this signaling pathway is compartmentalized in myocytes, as it was distinct from atrial natriuretic peptide receptor-cGMP-PKG-RyR2 Ser-2808 signaling and independent of muscarinic-induced phosphorylation of Ser-239 in vasodilator-stimulated phosphoprotein. These results provide detailed insights into muscarinic-induced PKG signaling and the mediators that regulate cardiac RyR2 phosphorylation critical for cardiovascular function.

O-Glycosylation of Camkii at Serine 280 Promotes Cardiac Arrhythmias in Diabetic Hyperglycemia

Diabetic cardiomyopathy is associated with increased arrhythmia risk. However, the exact molecular details in signaling and arrhythmia mechanisms remain incompletely understood. We measured action potentials (APs) in isolated murine ventricular myocytes exposed to high glucose (30 mM) versus control (5.5 mM glucose+24.5 mM mannitol to match osmolarity). Acute hyperglycemia prolonged AP duration, increased delayed afterdepolarizations and inducibility of AP-duration alternans during tachypacing. These effects were prevented by OSMI-1, a selective inhibitor of O-GlcNAc transferase (preventing O-GlcNAcylation), and enhanced by thiamet-G, a selective O-GlcNAcase inhibitor (that should promote O-GlcNAcylation). Thus, O-GlcNAcylation is necessary and sufficient for these arrhythmogenic alterations. These effects were also prevented in CaMKIIδ-knockout myocytes and in mice in which a known O-GlcNAcylated Serine on CaMKII was mutated (S280A knock-in). In streptozotocin-induced diabetes, myocytes bathed in normal (5.5 mM) glucose exhibited prolonged AP duration, enhanced delayed afterdepolarizations and alternans, all of which were worsened by acute hyperglycemia. Premature ventricular complexes on electrocardiograms were enhanced in diabetic WT but not in CaMKIIδ-knockout animals. Moreover, the arrhythmogenic remodeling in diabetes was abolished by the CaMKII inhibitor AIP or in S280A mice. We conclude that O-GlcNAcylation of CaMKII at Serine 280 enhances cardiac arrhythmias and represents a significant, novel therapeutic target in diabetes.

Optical Imaging of Isolated Murine Ventricular Myocytes

Optical Imaging of Isolated Murine Ventricular Myocytes

Optical Imaging of Isolated Murine Ventricular Myocytes.

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

The Antagonism of β3-AR and MICU1 Forms the Key Pathological Mechanism in Cardiac Hypertrophy

Mitochondrial calcium uptake 1 (MICU1) serves as a pivotal molecule in maintaining mitochondrial homeostasis, but its concrete role in cardiac hypertrophy remains elusive yet. The β3-adrenergic receptor (β3-AR) has received considerable attention of the world in cardiac hypertrophy, whether it possesses mutual restriction relation with MICU1 deserves further investigation. Specific small interfering RNAs against MICU1 or adenovirus-based plasmid with MICU1 were delivered into left ventricles or incubated with neonatal murine ventricular myocytes (NMVMs) for 48 h. Then, mice were rendered to cardiac hypertrophy by chronic infusion of angiotensin II (Ang-II) with a mini-osmotic pump for 28 days, meanwhile NMVMs were cultured with Ang-II for 48 h. Finally, MICU1 expression was downregulated in hypertrophic myocardium. MICU1 ablation aggravated cardiac hypertrophy both in vivo and in vitro, evidenced by impaired cardiac function, enlarged cardiomyocytes, increased hypertrophic maker genes and enhanced reactive oxygen species (ROS) generation. Moreover, excessive ROS accumulation following MICU1 deficiency increased β3-AR expression, which was reversed by N-acetyl-L-cysteine (NAC). In addition, β3-AR agonists BRL37344 depressed expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and MICU1, leading to aggravated cardiac hypertrophy. However, MICU1 supplementation eliminated those detrimental effects of BRL37344, and decreased the susceptibility of cardiomyocytes to hypertrophic stress. Furthermore, in hypertrophicmyocardium, melatonin alone, but not when combined with luzindole, inhibited β3-AR expression, accompanied with increased expression of PGC-1α/MICU1 and decreased generation of ROS, finally contributing to alleviated cardiac hypertrophy. Conclusively, the mutual antagonism of β3-AR and MICU1 gives rise to pathologic development of cardiac hypertrophy, and melatonin protects against hypertrophic stress by negative regulating the abovementioned pathway. Funding Statement: This work was supported by grants from National Science Funds of China (No.81500208; 81670419), Science Fund for Distinguished Young Scholars of Sichuan Province (No.2017JQ0012), China Postdoctoral Science Foundation (No.2017M613429). Declaration of Interests: The authors state: No competing financial interests exist. Ethics Approval Statement: All experiments performed in adherence to the National Institutes of Health Guidelines on the Use of Laboratory Animals (Bethesda, MD, USA) and conformed to the Institutional Animal Care and Use Committee of Chengdu Military General Hospital.

A protocol for registration and correction of multicolour STED superresolution images.

Multicolour fluorescence imaging by STimulated Emission Depletion (STED) superresolution microscopy with doughnut-shaped STED laser beams based on different wavelengths for each colour channel requires precise image registration. This is especially important when STED imaging is used for co-localisation studies of two or more native proteins in biological specimens to analyse nanometric subcellular spatial arrangements. We developed a robust postprocessing image registration protocol, with the aim to verify and ultimately optimise multicolour STED image quality. Importantly, this protocol will support any subsequent quantitative localisation analysis at nanometric scales. Henceforth, using an approach that registers each colour channel present during STED imaging individually, this protocol reliably corrects for optical aberrations and inadvertent sample drift. To achieve the latter goal, the protocol combines the experimental sample information, from corresponding STED and confocal images using the same optical beam path and setup, with that of an independent calibration sample. As a result, image registration is based on a strategy that maximises the cross-correlation between sequentially acquired images of the experimental sample, which are strategically combined by the protocol. We demonstrate the general applicability of the image registration protocol by co-staining of the ryanodine receptor calcium release channel in primary mouse cardiomyocytes. To validate this new approach, we identify user-friendly criteria, which - if fulfilled - support optimal image registration. In summary, we introduce a new method for image registration and rationally based postprocessing steps through a highly standardised protocol for multicolour STED imaging, which directly supports the reproducibility of protein co-localisation analyses. Although the reference protocol is discussed exemplarily for two-colour STED imaging, it can be readily expanded to three or more colours and STED channels.

Characterization of Intracellular Sodium Homeostasis in Murine Atrial Myocytes

Intracellular sodium concentration ([Na+]i) is an important regulator of intracellular Ca2+ and provides insight into the activation of the sarcolemmal sodium calcium exchanger (NCX) and the behavior of both Na+ channels and the Na+,K+-ATPase. In atrial diseases such as atrial fibrillation (AF), intracellular Ca2+ signaling is profoundly altered. While the mechanisms underlying altered intracellular Ca2+ homeostasis are well characterized, the role that [Na+]i may play and its dysregulation in disease in atrial myocytes is less well understood. For example, we have previously shown that high rate (10 Hz) atrial activation for 5 days in a rabbit model leads to an expected but significant reduction in [Na+]i (Greiser et al, JCI, 2014; 124: 4759-72).Here we characterize intracellular Na+ homeostasis in murine atrial myocytes using the Na+ indicator SBFI (Molecular Probes) in quiescent cells. Dual UV excitation (340 nm and 380 nm) was provided by a rapid switching Sutter DG5-plus illuminator using a 300 W xenon light source with widefield imaging by an EMCCD camera (Princeton Instruments). Freshly isolated atrial myocytes were loaded with 10 μmol/l SBFI-AM for 90 min in the presence of the non-ionic surfactant Pluronic F-127 (0.05 %w/v). After washout of the extra cellular dye, SBFI-AM de-esterification was allowed for 20 minutes before proceeding with [Na+]i measurements. Fluorescence images (F340 and F380) were collected at 510 ± 40 nm. The calibration of [Na+]i was performed in each cell separately after the measurements of quiescent [Na+]i by exposing the SBFI-loaded myocytes to various extracellular [Na+] ([Na+]o) in the presence of 10 μmol/l gramicidin D and 100 μmol/l-strophanthidin. Resting [Na+]i in mouse atrial myocytes was 8.2 ± 0.6 mM. External field stimulation (4 ms, 20 V, 1 Hz) increased [Na+]i by 3.1 ± 0.2 mM. These data show that [Na+]i in murine atrial myocytes is lower than in murine ventricular myocytes (12 ± 1 mM, Correll et al, Circ Res, 2014;114:249-56). These data and more will provide a broad understanding of atrial Na+ homeostasis and its implication for atrial myocyte Ca2+ signaling.

Melatonin attenuates postmyocardial infarction injury via increasing Tom70 expression

Mitochondrial dysfunction leads to reactive oxygen species (ROS) overload, exacerbating injury in myocardial infarction (MI). As a receptor for translocases in the outer mitochondrial membrane (Tom) complex, Tom70 has an unknown function in MI, including melatonin-induced protection against MI injury. We delivered specific small interfering RNAs against Tom70 or lentivirus vectors carrying Tom70a sequences into the left ventricles of mice or to cultured neonatal murine ventricular myocytes (NMVMs). At 48h post-transfection, the left anterior descending coronary arteries of mice were permanently ligated, while the NMVMs underwent continuous hypoxia. At 24h after ischemia/hypoxia, oxidative stress was assessed by dihydroethidium and lucigenin-enhanced luminescence, mitochondrial damage by transmission electron microscopy and ATP content, and cell apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling and caspase-3 assay. At 4weeks after ischemia, cardiac function and fibrosis were evaluated in mice by echocardiography and Masson's trichrome staining, respectively. Ischemic/hypoxic insult reduced Tom70 expression in cardiomyocytes. Tom70 downregulation aggravated post-MI injury, with increased mitochondrial fragmentation and ROS overload. In contrast, Tom70 upregulation alleviated post-MI injury, with improved mitochondrial integrity and decreased ROS production. PGC-1α/Tom70 expression in ischemic myocardium was increased with melatonin alone, but not when combined with luzindole. Melatonin attenuated post-MI injury in control but not in Tom70-deficient mice. N-acetylcysteine (NAC) reversed the adverse effects of Tom70 deficiency in mitochondria and cardiomyocytes, but at a much higher concentration than melatonin. Our findings showed that Tom70 is essential for melatonin-induced protection against post-MI injury, by breaking the cycle of mitochondrial impairment and ROS generation.

Adrenomedullin 2 activates extracellular-signal-regulated kinase in endothelial cells via a protein kinase C α-independent pathway

Adrenomedullin 2 plays diverse physiological roles such as regulating cardiovascular functions and blood pressure. It was reported that adrenomedullin 2 can activate protein kinase C in murine ventricular myocytes to augment cardiomyocyte contractile function. Using a protein kinase Cα knockout mouse model, we show here that adrenomedullin 2 activates extracellular-signal-regulated kinase in a protein kinase Cα-independent mechanism in endothelial cells.

NOS1 induces NADPH oxidases and impairs contraction kinetics in aged murine ventricular myocytes.

Nitric oxide (NO) modulates calcium transients and contraction of cardiomyocytes. However, it is largely unknown whether NO contributes also to alterations in the contractile function of cardiomyocytes during aging. Therefore, we analyzed the putative role of nitric oxide synthases and NO for the age-related alterations of cardiomyocyte contraction. We used C57BL/6 mice, nitric oxide synthase 1 (NOS1)-deficient mice (NOS1(-/-)) and mice with cardiomyocyte-specific NOS1-overexpression to analyze contractions, calcium transients (Indo-1 fluorescence), acto-myosin ATPase activity (malachite green assay), NADPH oxidase activity (lucigenin chemiluminescence) of isolated ventricular myocytes and cardiac gene expression (Western blots, qPCR). In C57BL/6 mice, cardiac expression of NOS1 was upregulated by aging. Since we found a negative regulation of NOS1 expression by cAMP in isolated cardiomyocytes, we suggest that reduced efficacy of β-adrenergic signaling that is evident in aged hearts promotes upregulation of NOS1. Shortening and relengthening of cardiomyocytes from aged C57BL/6 mice were decelerated, but were normalized by pharmacological inhibition of NOS1/NO. Cardiomyocytes from NOS1(-/-) mice displayed no age-related changes in contraction, calcium transients or acto-myosin ATPase activity. Aging increased cardiac expression of NADPH oxidase subunits NOX2 and NOX4 in C57BL/6 mice, but not in NOS1(-/-) mice. Similarly, cardiac expression of NOX2 and NOX4 was upregulated in a murine model with cardiomyocyte-specific overexpression of NOS1. We conclude that age-dependently upregulated NOS1, putatively via reduced efficacy of β-adrenergic signaling, induces NADPH oxidases. By increasing nitrosative and oxidative stress, both enzyme systems act synergistically to decelerate contraction of aged cardiomyocytes.

Impaired calcium-calmodulin-dependent inactivation of Cav1.2 contributes to loss of sarcoplasmic reticulum calcium release refractoriness in mice lacking calsequestrin 2

AimsIn cardiac muscle, Ca2+ release from sarcoplasmic reticulum (SR) is reduced with successively shorter coupling intervals of premature stimuli, a phenomenon known as SR Ca2+ release refractoriness. We recently reported that the SR luminal Ca2+ binding protein calsequestrin 2 (Casq2) contributes to release refractoriness in intact mouse hearts, but the underlying mechanisms remain unclear. Here, we further investigate the mechanisms responsible for physiological release refractoriness. Methods and resultsGene-targeted ablation of Casq2 (Casq2 KO) abolished SR Ca2+ release refractoriness in isolated mouse ventricular myocytes. Surprisingly, impaired Ca2+-dependent inactivation of L-type Ca2+ current (ICa), which is responsible for triggering SR Ca2+ release, significantly contributed to loss of Ca2+ release refractoriness in Casq2 KO myocytes. Recovery from Ca2+-dependent inactivation of ICa was significantly accelerated in Casq2 KO compared to wild-type (WT) myocytes. In contrast, voltage-dependent inactivation measured by using Ba2+ as charge carrier was not significantly different between WT and Casq2 KO myocytes. Ca2+-dependent inactivation of ICa was normalized by intracellular dialysis of excess apo-CaM (20μM), which also partially restored physiological Ca2+ release refractoriness in Casq2 KO myocytes. ConclusionsOur findings reveal that the intra-SR protein Casq2 is largely responsible for the phenomenon of SR Ca2+ release refractoriness in murine ventricular myocytes. We also report a novel mechanism of impaired Ca2+-CaM-dependent inactivation of Cav1.2, which contributes to the loss of SR Ca2+ release refractoriness in the Casq2 KO mouse model and, therefore, may further increase risk for ventricular arrhythmia in vivo.

Loss of PI3K-Gamma Scaffold Function causes Severe Electrical Remodeling in Mice Ventricular Myocytes

In the heart the kinase/scaffold protein phosphoinositide-3-kinase-γ (PI3Kγ) has been associated with maladaptive remodeling. However little is known about the relative contribution of kinase-scaffold roles on the normal electrical activity in cardiac cells.Here, we compared the action potentials and ionic currents in ventricular myocytes isolated from conventional KO mice or mice expressing a catalytically inactive PI3K (“kinase-dead”, KD). Action potentials were evoked at 1Hz (I-clamp). K+ and L-type Ca2+ (ICaL) currents were measured by step depolarizations (V-clamp).Membrane capacitances (Cm) were not different between genotypes. KO myocytes showed marked prolongation of the action potential duration (APD) at 90% of repolarization (APD90: WT 64.36±4.1 ms, KO 149.6±18 ms). APD90 beat-to-beat variability (BVR) was larger in KO and, consistently, the incidence of early afterdepolarizations was higher in KO than WT (respectively, 66% and 18%). In contrast, KD cells showed a slight prolongation of APD90 (96±7.9 ms), but BVR and EADs incidence were comparable to WT. While voltage-gated K+ currents (Ito, IKur and Iss) were strongly downregulated in KO, KD myocytes showed only a small reduction of Ito. ICaL density was significantly lower in both KO and KD mice at negative potentials only. Consistently, both KD and KO mice showed a positive shift of both steady-state activation (ΔV1/2 vs WT: KO 4.2 mV, KD 5.5 mV) and inactivation curves (ΔV1/2 vs WT: KO 3.2 mV, KD 3.4 mV). Addition of isoproterenol (100 nM) resulted in ICaL enhancement, whose magnitude was larger in KO compared to KD and WT myocytes (% increase,WT: 58±6%,KD:45±7%,KO:95.5±12%). These data suggest distinct action related to either kinase and scaffold functions: while the former has an impact on ICaL biophysical properties, the latter prevents arrhythmias in murine ventricular myocytes by preserving K+ currents.

In vivo Temperature Sensitivity of the Calcium Affinity of fluo-5F and mag-fluo4

The role of calcium ion (Ca2+) in diverse cellular signaling pathways has been identified and investigated principally by using fluorescent chelators as indicators. Using the biophysical characteristics of a fluorescent Ca2+ indicator allows investigators to convert measured fluorescence intensity into values of free Ca2+ concentration ([Ca2+]). Troublingly, the characteristics of Ca2+-indicators are dependent on numerous environmental factors. Determining the behavior of each indicator in vivo under comparable experimental conditions is necessary for quantitative measurements. Temperature is one environmental factor that strongly affects Ca2+ indicator behavior. A change in temperature can change the intrinsic physical characteristics of the indicator (e.g., Ca2+ affinity, quantum efficiency, and fluorescence lifetime), as well as change the cellular processes that affect indicator performance (e.g., extrusion by transporters, cellular pH). Therefore, experiments were designed to estimate the disassociation constants (KD) of the Ca2+ indicators fluo-5F and mag-fluo4 (loaded into the cytosol and sarcoplasmic reticulum, respectively) at room and physiological temperatures in murine ventricular myocytes. Ca2+ access to cytosolic compartment was established using a Ca2+ ionophore, while sarcoplasmic reticulum (SR) access was established by sarcolemma permeabilization with saponin in the presence of caffeine. Once access was established, buffers with known [Ca2+] were rapidly applied using a micro-perfusion system with temperature feedback control. We observed that, increasing from room temperature to 36°C caused a modest decrease in the KD of both fluo-5F and mag-fluo4. Both indicators also displayed a decrease in dynamic range when heated. Additionally, temperature-dependent extrusion was observed for indicators loaded into the cytosol, but not those loaded into the SR. These findings are important in enabling quantitative interpretation of measurements in living cells under physiological conditions.