Carbon monoxide levels experienced by heavy smokers impair aerobic capacity and cardiac contractility and induce pathological hypertrophy

Cigarette smoke contains hundreds of potentially toxic compounds and is an important risk factor for cardiovascular disease. However, the key components responsible for endothelial and myocardial dysfunction have not been fully identified. The objective of the present study was to determine the cardiovascular effects of long-term inhalation of carbon monoxide (CO) administrated to give concentrations in the blood similar to those observed in heavy smokers. Female rats were exposed to either CO or air (control group) (n = 12). The CO group was exposed to 200 ppm CO (100 h/wk) for 18 mo. Rats exposed to CO had 24% lower maximal oxygen uptake, longer (145 vs. 123 microm) and wider (47 vs. 25 microm) cardiomyocytes, reduced cardiomyocyte fractional shortening (12 vs. 7%), and 26% longer time to 50% re-lengthening than controls. In addition, cardiomyocytes from CO-exposed rats had 48% lower intracellular calcium (Ca2 +) amplitude, 22% longer time to Ca2 + decay, 34% lower capacity of sarcoplasmic reticulum Ca2 +-ATPase (SERCA2a), and 37% less t-tubule area compared to controls. Phosphorylation levels of phospholamban at Ser16 and Thr17 were significantly reduced in the CO group, whereas total concentration of phospholamban and SERCA2a were unchanged. Cardiac atrial natriuretic peptide, vascular endothelial growth factor, cyclic guanosine monophosphate, calcineurin, calmodulin, pERK, and pS6 increased, whereas pAkt and pCaMKII delta remained unchanged by CO. Endothelial function and systemic blood pressure were not affected by CO exposure. Long-term CO exposure reduces aerobe capacity and contractile function and leads to pathological hypertrophy. Impaired Ca2 + handling and increased growth factor signaling seem to be responsible for these pathological changes.     

nNOS抑制剂亚胺基烯丁基-L-鸟氨酸对心肌缺血再灌注损伤的影响及机制

目的 探讨nNOS选择性抑制剂亚胺基烯丁基-L-鸟氨酸（L-VNIO）对心肌缺血再灌注（I/R）损伤的影响及机制。方法 构建SD大鼠离体心脏I/R模型和H9c2细胞缺氧/复氧（H/R）模型；nNOS抑制剂L-VNIO（10 μmol·L^-1）持续给药整个再灌注或复氧过程。TTC染色测定心肌梗死面积；流式细胞术检测H9c2细胞凋亡率；Fluo-3/AM Ca²⁺荧光探针通过流式细胞仪检测H9c2细胞内Ca²⁺浓度；试剂盒法测定离体心脏灌流液乳酸脱氢酶（LDH）、丙二醛（MDA）水平以及H9c2细胞MDA水平和超氧化物歧化酶（SOD）活性；离体心脏提取肌浆网，试剂盒法检测肌浆网Ca²⁺-ATP酶（SERCA）活性，Western blotting检测肌浆网SERCA蛋白表达；Western blotting检测离体心脏中受磷蛋白（PLB）和兰尼碱受体2（RyR2）蛋白表达水平和磷酸化水平。结果 与I/R或H/R模型组相比，L-VNIO显著降低细胞凋亡率，减少心肌梗死面积，降低LDH、MDA水平，提高SOD活性，差异均有统计学意义（P<0.05）；此外，与I/R或H/R模型组相比，L-VNIO组明显降低细胞内Ca²⁺超载，增高PLB磷酸化水平，降低RyR2磷酸化水平，增强SERCA活性（P<0.05）。结论 nNOS抑制剂L-VNIO可以减轻I/R损伤，机制与调节Ca²⁺转运相关蛋白而降低I/R引起的Ca²⁺超载相关。     

SERCA2 activity is involved in the CNP-mediated functional responses in failing rat myocardium

BACKGROUND AND PURPOSES

Myocardial C-type natriuretic peptide (CNP) levels are increased in heart failure. CNP can induce negative inotropic (NIR) and positive lusitropic responses (LR) in normal hearts, but its effects in failing hearts are not known. We studied the mechanism of CNP-induced NIR and LR in failing hearts and determined whether sarcoplasmatic reticulum Ca²⁺ ATPase2 (SERCA2) activity is essential for these responses.

EXPERIMENTAL APPROACH

Contractility, cGMP levels, Ca²⁺ transient amplitudes and protein phosphorylation were measured in left ventricular muscle strips or ventricular cardiomyocytes from failing hearts of Wistar rats 6 weeks after myocardial infarction.

KEY RESULTS

CNP increased cGMP levels, evoked a NIR and LR in muscle strips, and caused phospholamban (PLB) Ser¹⁶ and troponin I (TnI) Ser^23/24 phosphorylation in cardiomyocytes. Both the NIR and LR induced by CNP were reduced in the presence of a PKG blocker/cGMP analogue (Rp-8-Br-Pet-cGMPS) and the SERCA inhibitor thapsigargin. CNP increased the amplitude of the Ca²⁺ transient and increased SERCA2 activity in cardiomyocytes. The CNP-elicited NIR and LR were not affected by the L-type Ca²⁺ channel activator BAY-K8644, but were abolished in the presence of isoprenaline (induces maximal activation of cAMP pathway). This suggests that phosphorylation of PLB and TnI by CNP causes both a NIR and LR. The NIR to CNP in mouse heart was abolished 8 weeks after cardiomyocyte-specific inactivation of the SERCA2 gene.

CONCLUSIONS AND IMPLICATIONS

We conclude that CNP-induced PLB and TnI phosphorylation by PKG in concert mediate both a predictable LR as well as the less expected NIR in failing hearts.     

Imbalance between CaM kinase II and calcineurin activities impairs caffeine-induced calcium release in hypertrophic cardiomyocytes

Cardiac hypertrophy impairs Ca²⁺ handling in the sarcoplasmic reticulum, thereby impairing cardiac contraction. To identify the mechanisms underlying impaired Ca²⁺ release from the sarcoplasmic reticulum in hypertrophic cardiomyocytes, we assessed Ca²⁺-dependent signaling and the phosphorylation of phospholamban, which regulates Ca²⁺ uptake during myocardial relaxation and is in turn regulated by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and calcineurin. In cultured rat cardiomyocytes, treatment with endothelin-1, angiotensin II, and phenylephrine-induced hypertrophy and increased CaMKII autophosphorylation and calcineurin expression. The calcineurin level reached its maximum at 72 h and remained elevated for at least 96 h after endothelin-1 or angiotensin II treatment. By contrast, CaMKII autophosphorylation, phospholamban phosphorylation, and caffeine-induced Ca²⁺ mobilization all peaked 48 h after these treatments. By 96 h after treatment, CaMKII autophosphorylation and phospholamban phosphorylation had returned to baseline, and caffeine-induced Ca²⁺ mobilization was impaired relative to baseline. A similar biphasic change was observed in dystrophin levels in endothelin-1-induced hypertrophic cardiomyocytes, and treatment with the novel CaM antagonists DY-9760e and DY-9836 significantly inhibited the hypertrophy-induced dystrophin breakdown. Taken together, the abnormal Ca²⁺ regulation in cardiomyocytes following hypertrophy is in part mediated by an imbalance in calcineurin and CaMKII activities, which leads to abnormal phospholamban activity.     

Acute methamphetamine exposure inhibits cardiac contractile function

Methamphetamine, a commonly seen substance of abuse, has been reported to exert detrimental effect on bodily function including the cardiovascular system although its mechanism of action is poorly understood. This study was designed to examine the direct impact of methamphetamine on isolated whole heart and single cardiomyocyte contractile function. Murine hearts and isolated cardiomyocytes from adult FVB mice were exposed to various concentrations of methamphetamine for 30 min prior to the assessment of mechanical function using a Langendroff apparatus and an IonOptix Myocam^® system, respectively. Cardiac contractile properties analyzed included maximal velocity of left ventricular pressure development and decline (±dP/dt), peak shortening amplitude (PS), maximal velocity of shortening/relengthening (±dLdt), time-to-PS (TPS), time-to-90% relengthening (TR₉₀), resting and electrically stimulated increase of intracellular Ca²⁺ as well as intracellular Ca²⁺ decay. Our results revealed that acute methamphetamine exposure depressed ±dP/dt, PS and rise of intracellular Ca²⁺ without affecting ±dLdt, TPS, TR₉₀, resting intracellular Ca²⁺ and intracellular Ca²⁺ decay. Furthermore, methamphetamine nullified the adrenergic agonist norepinephrine-elicited positive cardiomyocyte contractile response, including elevated PS, ±dLdt and shortened TR₉₀ without affecting TPS. Western blot analysis showed unchanged expression of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2a) and phospholamban, associated with upregulated Na⁺–Ca²⁺ exchanger levels following acute methamphetamine exposure. In addition, methamphetamine promoted overt cardiomyocyte protein damage evaluated by carbonyl formation. Taken together, these results demonstrate direct cardiac depressant effect of methamphetamine in myocardium and isolated cardiomyocytes, possibly associated with protein damage and dampened adrenergic response.     

Insulin-like growth factor I (IGF-1) deficiency ameliorates sex difference in cardiac contractile function and intracellular Ca homeostasis

Sex difference in cardiac contractile function exists which may contribute to the different prevalence in cardiovascular diseases between genders. However, the precise mechanisms of action behind sex difference in cardiac function are still elusive. Given that sex difference exists in insulin-like growth factor I (IGF-1) cascade, this study is designed to evaluate the impact of severe liver IGF-1 deficiency (LID) on sex difference in cardiac function. Echocardiographic, cardiomyocyte contractile and intracellular Ca²⁺ properties were evaluated including ventricular geometry, fractional shortening, peak shortening, maximal velocity of shortening/relengthening (±dL/dt), time-to-peak shortening (TPS), time-to-90% relengthening (TR₉₀), fura-fluorescence intensity (FFI) and intracellular Ca²⁺ clearance. Female C57 mice exhibited significantly higher plasma IGF-1 levels than their male counterpart. LID mice possessed comparably low IGF-1 levels in both sexes. Female C57 and LID mice displayed lower body, heart and liver weights compared to male counterparts. Echocardiographic analysis revealed larger LV mass in female C57 but not LID mice without sex difference in other cardiac geometric indices. Myocytes from female C57 mice exhibited reduced peak shortening, ±dL/dt, longer TPS, TR₉₀ and intracellular Ca²⁺ clearance compared with males. Interestingly, this sex difference was greatly attenuated or abolished by IGF-1 deficiency. Female C57 mice displayed significantly decreased mRNA and protein levels of Na⁺-Ca²⁺ exchanger, SERCA2a and phosphorylated phospholamban as well as SERCA activity compared with male C57 mice. These sex differences in Ca²⁺ regulatory proteins were abolished or overtly attenuated by IGF-1 deficiency. In summary, our data suggested that IGF-1 deficiency may significantly attenuated or mitigate the sex difference in cardiomyocyte contractile function associated with intracellular Ca²⁺ regulation.     

Dissection of the Effects of Quercetin on Mouse Myocardium

Quercetin is a plant flavonoid with several biological activities. This study aimed to describe quercetin effects on contractile and electrophysiological properties of the cardiac muscle as well as on calcium handling. Quercetin elicited positive inotropism that was significantly reduced by propranolol indicating an involvement of the sympathetic nervous system. In cardiomyocytes, 30 μM quercetin increased I_Ca,L at 0 mV from ?0.95 ± 0.01 A/F to ?1.21 ± 0.08 A/F. The membrane potential at which 50% of the channels are activated (V_0.5) shifted towards more negative potentials from ?13.06 ± 1.52 mV to ?19.26 ± 1.72 mV and did not alter the slope factor. Furthermore, quercetin increased [Ca²⁺]_i transient by 28% when compared to control. Quercetin accelerated [Ca²⁺]_i transient decay time, which could be attributed to SERCA activation. In resting cardiomyocytes, quercetin did not change amplitude or frequency of Ca²⁺ sparks. In isolated heart, quercetin increased heart rate and decreased PRi, QTc and duration of the QRS complex. Thus, we showed that quercetin activates β‐adrenoceptors, leading to increased L‐type Ca²⁺ current and cell‐wide intracellular Ca²⁺ transient without visible changes in Ca²⁺ sparks.     

升陷汤及单味药材水提物对缺氧/复氧致心肌损伤的保护作用

目的 通过观察升陷汤及单味药材水提物对离体培养的大鼠心肌细胞缺氧/复氧损伤的影响,并对其作用机制进行初步探讨。方法 培养H9C2大鼠心肌细胞,共分成8组：空白对照组,缺氧/复氧组（模型组）,缺氧复氧损伤后药物干预组（升陷汤全方及5个单味药材水提物组）。分别对心肌细胞凋亡率、心肌细胞的活力、细胞内活性氧（ROS）活性、细胞内钙离子浓度（Ca²⁺）等指标进行检测。结果 升陷汤全方及黄芪、知母等药材干预能明显降低细胞凋亡率、细胞内ROS活性和Ca²⁺浓度（P<0.05）,其中,全方的作用最强。与缺氧/复氧组细胞内ROS活性和Ca²⁺浓度增加至空白对照组的2.49倍及1.71倍相比,全方能使细胞内ROS活性和Ca²⁺浓度增加率降至缺氧/复氧组的41.37%和15.20%。结论 升陷汤及单味药材对缺氧/复氧致心肌损伤具有保护作用,该作用的机制可能通过抑制细胞凋亡、降低细胞内ROS以及Ca²⁺的浓度所致。     

Thaliporphine ameliorates cardiac depression in endotoxemic rats through attenuating TLR4 signaling in the downstream of TAK-1 phosphorylation and NF-κB signaling

Thaliporphine was found to ameliorate endotoxin-induced circulatory failure and mortality in rodents. The aims of the present study were to assess whether thaliporphine could improve cardiac function in endotoxemic rats and to investigate the underlying mechanisms. Cardiac function was evaluated by pressure–volume loop analysis in pentobarbital-anesthetized rats 24 h after intravenous injection of lipopolysaccharide (LPS) (4 mg/kg) with or without thaliporphine (1 mg/kg, iv). The intracellular Ca²⁺ transients, nitric oxide (NO), and reactive oxygen species (ROS) in enzymatically isolated ventricular cells were measured by fluorescent indicators. Western blotting was used to analyze the change of protein expression in response to LPS with or without thaliporphine in rat ventricle, H9C2 and Raw264.7 cells. Cardiac depression was found to coincide with the decreased intracellular Ca²⁺ transients and the increased expression of nitrotyrosine on SERCA2 in rat ventricles after 24-h endotoxemia. Thaliporphine decreased intracellular NO and ROS level in ventricular cells and the nitrosylation of SERCA2, which resulted in recovering the functional properties of intracellular Ca²⁺ handling and cardiac contraction. In H9C2 cells, LPS-induced nuclear translocation of nuclear factor kappa B (NF-κB) could be attenuated by thaliporphine. In Raw264.7 cells, thaliporphine attenuated LPS-induced TAK-1 phosphorylation and IκBα degradation in association with the inhibition of inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNF-α) expression and the production of NO and ROS. In conclusion, thaliporphine ameliorates LPS-induced cardiac depression through attenuating TLR4 signaling in the downstream of TAK-1 phosphorylation and NF-κB signaling in both cardiomyocytes and macrophage to prevent cardiac SERCA2 from nitrosylation by peroxynitrite via decreasing iNOS and TNF-α expression.     

Oestrogen upregulates the sarcoplasmic reticulum Ca2+ ATPase pump in coronary arteries

The presence of circulating plasma 17β‐oestradiol (E2) is beneficial in women against abnormal vascular tone development, such as coronary arterial vasospasms. Several vascular diseases have demonstrated that increased expression of the sarcoplasmic reticulum Ca²⁺‐ATPase pump (SERCA2b) serves to limit the excessive accumulation of intracellular Ca²⁺. Therefore, the hypothesis of the present study was that E2 would increase SERCA2b expression in the coronary vasculature. Coronary arteries were dissected from hearts obtained from mature female pigs. Artery segments were cultured for 24 h in E2 (1 pmol/L or 1 nmol/L) and homogenized for western blot analysis. At 1 nmol/L, E2 induced an approximate 50% increase in immunoreactivity for SERCA2b. In addition, E2 increased the protein expression of the known SERCA regulatory proteins, protein kinase A (PKA) and protein kinase G (PKG). The E2‐induced increase in SERCA2b was attenuated when the culture medium was supplemented with the oestrogen receptor (ER) α/β antagonist ICI 182,780 and the PKG antagonist KT5823 (10 μmol/L, 24 h for both). The PKA antagonist (KT5720; 10 μmol/L, 24 h) had no effect on SERCA2b expression. Removal of the endothelium (using a wooden toothpick) from artery segments prior to culture decreased the E2‐mediated increase in SERCA2b and PKG expression by 45% and 47%, respectively. Overall, the findings suggest that one of the potential cardiovascular benefits of E2 in women is upregulation of SERCA2b, via activation of the classic ERα and ERβ pathway.     

Effect of heparin treatment on the expression and activity of different ion-motive P-type ATPase isoforms from mouse extensor digitorum longus muscle during degeneration and regeneration after Bothrops jararacussu venom injection

Ca²⁺ ions are essential to myonecrosis, a serious complication of snake envenomation, and heparin seems to counteract this effect. We investigated the effect of local injection of Bothrops jararacussu venom in mouse fast-twitch extensor digitorum longus (EDL) muscle, without or with heparin, on functional/molecular alterations of two central proteins involved in intracellular Ca²⁺ homeostasis, sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) and Na⁺/K⁺-ATPase. EDL-specific SERCA1 isoform expression dropped significantly just after venom administration (up to 60% compared to control EDL values at days 1 and 3; p < 0.05) while SERCA2 and Na⁺/K⁺-ATPase α₁ isoform expression increased at the same time (3-6- and 2-3-fold, respectively; p < 0.05). Although not significant, Na⁺/K⁺-ATPase α₂ isoform followed the same trend. Except for SERCA2, all proteins reached basal levels at the 7th day. Intravenous heparin treatment did not affect these profiles. Ca²⁺-ATPase activity was also decreased during the first days after venom injection, but here heparin was effective to reinstate activity to control levels within 3 days. We also showed that B. jararacussu venom directly inhibited Ca²⁺-ATPase activity in a concentration-dependent manner. Our results indicate that EDL SERCA and Na⁺/K⁺-ATPase are importantly affected by B. jararacussu venom and heparin has protective effect on activity but not on protein expression.     

Commonly used ryanodine receptor activator, 4-chloro-m-cresol (4CmC), is also an inhibitor of SERCA Ca2+ pumps

4-Chloro-m-cresol (4CmC) is an extensively used activator of ryanodine receptors (RyRs). Studies have shown that 4CmC, at a concentration of 1 mM, is sufficient to cause Ca²⁺ release through RyRs. Here, we show that mM concentrations of 4CmC also inhibit the sarcoplasmic-endoplasmic reticulum Ca²⁺-ATPase (SERCA), (IC₅₀ 2–3mM) and cause Ca²⁺ release. 4CmC also causes increased intracellular [Ca²⁺] levels in COS-7 cells, which lack functional RyRs. Thus, any increase in [Ca²⁺] levels associated with use of 4CmC (≥ 1mM) could lead to non-specific Ca²⁺ changes due to SERCA inhibition rather than RyR activation.     

ENDOTHELIUM-SMOOTH MUSCLE INTERACTIONS IN BLOOD VESSELS

1. Blood vessel tone is determined both by smooth muscle and endothelial functions. In coronary arteries taken from rat (Fisher-Lewis) cardiac transplanted hearts, the inducible form of NOS (iNOS) in smooth muscle is more active, while acetylcholine-induced nitric oxide production in the endothelium is greatly diminished. This causes a greatly reduced myogenic constriction, in pressurized septal arteries taken from immunologically challenged transplanted hearts. 2. The sarcoplasmic reticulum (SR) of smooth muscle and the endoplasmic reticulum (ER) of endothelial cells sequeste. Ca²⁺ from the cytoplasm. This reduces the intracellular concentration of free Ca²⁺, which is necessary for the activation of cellular processes. The release of Ca²⁺ from internal stores occurs through ryanodine and IP3 recoptors located on the SR membrane. 3. The superficial SR/ER also interacts with ion exchangers and pumps in the plasma membrane. This allows for the superficial SR/ER to function in Ca²⁺ extrusion; for example, inhibition of the SR/ER Ca²⁺-ATPase (SERCA) partially inhibits the rate of loss Ca²⁺ from the cell. Recent data suggest that the SR Ca²⁺-ATPase and the Na⁺-Ca²⁺ exchanger of smooth muscle cells function in series; that is, Ca²⁺ uptake by the SR followed by release towards the exchanger to mediate extrusion. This interaction between the SERCA of the superficial SR and ion exchangers and pumps creates intracellular Ca²⁺ gradients. 4. The SERCA of the superficial, peripherally distributed SR/ER also serves to regulat. Ca²⁺ entry from the extracellular space. This occurs in part by inhibition of the superficial buffer barrier function of the SR as well as by depletion of stimulated Ca²⁺ entry. 5. Ca²⁺ entry is also regulated in endothelial and smooth muscle cells by the membrane potential. Membrane hyperpolar-ization increases the driving force for Ca²⁺ entry into endothelial cells, which lack voltage-gated Ca²⁺ channels, and reduces open ate probability of voltage-gated Ca²⁺ channels in vascular smooth muscle cells. The two cell types have electrical contact and interact in a dynamic manner to regulate blood vessel diameter     

环维黄杨星D升高心肌细胞内游离钙浓度实验研究

观察了环维黄杨星D（CVB－D）对急性分离心肌细胞内游离Ca^2 浓度的影响。应用特异性荧光指示剂Fluo-3/AM负载细胞,用激光共聚焦显微镜检测胞内游离钙的变化。结果表明,CVB－D在10^-5,10^-4mol/L时均可升高心肌细胞内游离钙离子浓度,此作用随剂量增加而增加。因此,CVB－D具有升高心肌细胞内游离钙离子浓度的作用。     

Effects of Caulophine on Caffeine‐induced Cellular Injury and Calcium Homeostasis in Rat Cardiomyocytes

Abstract: Caulophine is a novel fluorenone alkaloid isolated from the radix of Caulophyllum robustum Maxim. Caulophine showed high affinity for the rat myocardial cell membrane as assessed by cell membrane chromatography, suggesting that the compound may exert bioactivity in the heart. It is known that calcium plays an important role in the pathogenesis of ischaemic heart disease, and caffeine can cause calcium overload in cardiomyocytes by inducing calcium release from the sarcoplasmic reticulum. Therefore, the present study evaluated the effects of caulophine on caffeine‐induced injury and calcium homeostasis in cardiomyocytes. Cardiomyocytes were pre‐treated with caulophine before exposure to caffeine or potassium chloride (KCl). Cell viability was assayed using the MTT method, and lactate dehydrogenase (LDH) and malondialdehyde (MDA) were measured spectrophotometrically. Caulophine‐pre‐treated cardiomyocytes were incubated with Fluo‐3/AM, and then caffeine or KCl was used to induce Ca²⁺ overload. The total intracellular Ca²⁺ concentration was measured by flow cytometry. Fluorescence densities of single cardiomyocytes were detected using a confocal microscope. Caulophine increased the viability of caffeine‐injured cardiomyocytes and decreased LDH activity and MDA level in cardiomyocytes. Furthermore, caulophine significantly decreased the total intracellular free Ca²⁺ concentration and intracellular calcium release in cardiomyocytes in response to caffeine. However, the same concentrations of caulophine did not affect KCl‐induced calcium influx. Our results suggest that caulophine protects cardiomyocytes from caffeine‐induced injury as a result of calcium antagonism. This finding provides a basis for further study and development of caulophine as a new calcium antagonist for treating ischaemic cardiovascular diseases.     

Cardiac adenovirus-associated viral Presenilin 1 gene delivery protects the left ventricular function of the heart via regulating RyR2 function in post-ischaemic heart failure

Post-ischaemic heart failure is a major cause of death worldwide. Reperfusion of infarcted heart tissue after myocardial infarction has been an important medical intervention to improve outcomes. However, disturbances in Ca²⁺ and redox homeostasis at the cellular level caused by ischaemia/reperfusion remain major clinical challenges. In this study, we investigated the potential of adeno-associated virus (AAV)-9-mediated cardiac expression of a Type-2 ryanodine receptor (RyR2) degradation-associated gene, Presenilin 1 (PSEN1), to combat post-ischaemic heart failure. Adeno-associated viral PSEN1 gene delivery elevated PSEN1 protein expression in a post-infarction rat heart failure model, and this administration normalised the contractile dysfunction of the failing myocardium in vivo and in vitro by reversing myocardial Ca²⁺ handling and function. Moreover, PSEN1 gene transfer to failing cardiomyocytes reduced sarcoplasmic reticulum (SR) Ca²⁺ leak, thereby restoring the diminished intracellular Ca²⁺ transients and SR Ca²⁺ load. Moreover, PSEN1 gene transfer reversed the phosphorylation of RyR2 in failing cardiomyocytes. However, selective autophagy inhibition did not prevent the PSEN1-induced blockade of RyR2 degradation, making the participation of autophagy in PSEN1-associated RyR2 degradation unlikely. Our results established a role of the cardiac expression of PSEN1 with AAV9 vectors as a promising therapeutic approach for post-ischaemic heart failure.     

Uncoupling of sarcoplasmic reticulum Ca²⁺-ATPase by N-arachidonoyl dopamine. Members of the endocannabinoid family as thermogenic drugs

BACKGROUND AND PURPOSE

The sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) plays a role in thermogenesis. The exogenous compound capsaicin increased SERCA-mediated ATP hydrolysis not coupled to Ca²⁺ transport. Here, we have sought to identify endogenous compounds that may function as SERCA uncoupling agents.

EXPERIMENTAL APPROACH

Using isolated SR vesicles from rabbits, we have screened for endogenous compounds that uncouple SERCA. We have also studied their ability to deplete cytoplasmic ATP from human skeletal muscle cells in culture.

KEY RESULTS

Studies on SR vesicles showed that the endogenous lipid metabolite N-arachidonoyl dopamine (NADA) was a potent stimulator of SERCA uncoupling. NADA stabilized an E₁-like pump conformation that had a lower dephosphorylation rate, low affinity for Ca²⁺ at the luminal sites and a specific proteinase K cleavage pattern involving protection of the C-terminal p83C fragment from further cleavage. Moreover, we found a significantly decreased cytoplasmic ATP levels following treatment of skeletal muscle cells with 100 nM NADA. This effect was dependent on the presence of glucose and abolished by pretreatment with the specific SERCA inhibitor thapsigargin, regardless of the presence of glucose.

CONCLUSIONS AND IMPLICATIONS

NADA is an endogenous molecule that may function as SERCA uncoupling agent in vivo. Members of the endocannabinoid family exert concerted actions on several Ca²⁺-handling proteins. Uncoupling of SERCA by exogenous compounds could be a novel post-mitochondrial strategy for reduction of cellular ATP levels. In addition, signalling networks leading to SERCA uncoupling can be explored to study the importance of this ion pump in pathophysiological conditions related to metabolism.     

Glycine inhibits the LPS-induced increase in cytosolic Ca2+ concentration and TNFα production in cardiomyocytes by activating a glycine receptor

Aim:

Previous studies have demonstrated that glycine (GLY) markedly reduces lipopolysaccharide (LPS)-induced myocardial injury. However, the mechanism of this effect is still unclear. The present study investigated the effect of GLY on cytosolic calcium concentration ([Ca²⁺]_c) and tumor necrosis factor-α (TNFα) production in cardiomyocytes exposed to LPS, as well as whether the glycine-gated chloride channel is involved in this process.

Methods:

Neonatal rat cardiomyocytes were isolated, and the [Ca²⁺]_c and TNFα levels were determined by using Fura-2 and a Quantikine enzyme-linked immunosorbent assay, respectively. The distribution of the GLY receptor and GLY-induced currents in cardiomyocytes were also investigated using immunocytochemistry and the whole-cell patch-clamp technique, respectively.

Results:

LPS at concentrations ranging from 10 ng/mL to 100 μg/mL significantly stimulated TNFα production. GLY did not inhibit TNFα production induced by LPS at concentrations below 10 ng/mL but did significantly decrease TNFα release stimulated by 100 μg/mL LPS and prevented an LPS-induced increase in [Ca²⁺]_c, which was reversed by strychnine, a glycine receptor antagonist. GLY did not block the isoproterenol-induced increase in [Ca²⁺]_c, but did prevent the potassium chloride-induced increase in [Ca²⁺]_c in cardiomyocytes. Strychnine reversed the inhibition of the KCl–stimulated elevation in [Ca²⁺]_c by GLY. In chloride-free buffer, GLY had no effect on the dipotassium hydrogen phosphate-induced increase in [Ca²⁺]_c. Furthermore, GLY receptor α₁ and β subunit-immunoreactive spots were observed in cardiomyocytes, and GLY-evoked currents were blocked by strychnine.

Conclusion:

Cardiomyocytes possess the glycine-gated chloride channel, through which GLY prevents the increase in [Ca²⁺]_c and inhibits the TNFα production induced by LPS at high doses in neonatal rat cardiomyocytes.     

17-β estradiol attenuates ovariectomy-induced changes in cardiomyocyte contractile function via activation of AMP-activated protein kinase

Menopause increases the risk of cardiometabolic diseases in women. This circumstance is usually attributed to a deficiency in circulating estrogen levels although the underlying mechanism remains elusive. Given the pivotal role of AMP-activated protein kinase (AMPK) in the regulation of energy metabolism and cardiac function, this study was designed to examine the role of AMPK in estrogen deficiency and replacement-exerted cardiomyocyte responses. Adult female WT and AMPK kinase dead (KD) mice were subjected to bilateral ovariectomy (OVX) or sham operation. A cohort of ovariectomized mice received 17β-estradiol (E2) (40 μg/kg/day, i.p.) for 6 weeks. Mechanical and intracellular Ca²⁺ properties were evaluated including peak shortening (PS), time-to-PS (TPS), time-to-90%-relengthening (TR₉₀), and maximal velocity of shortening/relengthening (±dL/dt). Levels of AMPK, Akt JNK, ACC, SERCA, membrane Glut4, AS160 and PGC-1α were assessed using Western blot. OVX significantly decreased PS, ±dL/dt and intracellular Ca²⁺ rise in responsible to electric stimulus, prolonged TR₉₀ and intracellular Ca²⁺ decay without affecting TPS and resting intracellular Ca²⁺, the effects of which were reconciled by E2 replacement. Western blot analysis depicted that OVX suppressed phosphorylation of Akt AMPK and ACC although it promoted JNK phosphorylation, the effects of which were mitigated or significantly attenuated by E2 treatment in WT but not KD mice. Moreover, OVX procedure downregulated SERCA2a and membrane Glut4 while inhibiting AS160 phosphorylation without affecting PGC-1α levels. In vitro study revealed that E2 corrected cardiomyocyte contractile dysfunction elicited by OVX in cardiomyocytes from WT but not the AMPK kinase dead mice. Taken together, these data suggest that E2 treatment ameliorates estrogen deficiency-induced changes in cardiac contractile function possibly through an AMPK-dependent mechanism.