David A. MacDougall

Caveolae compartmentalise β2-adrenoceptor signals by curtailing cAMP production and maintaining phosphatase activity in the sarcoplasmic reticulum of the adult ventricular myocyte

Inotropy and lusitropy in the ventricular myocyte can be efficiently induced by activation of β1-, but not β2-, adrenoceptors (ARs). Compartmentation of β2-AR-derived cAMP-dependent signalling underlies this functional discrepancy. Here we investigate the mechanism by which caveolae (specialised sarcolemmal invaginations rich in cholesterol and caveolin-3) contribute to compartmentation in the adult rat ventricular myocyte. Selective activation of β2-ARs (with zinterol/CGP20712A) produced little contractile response in control cells but pronounced inotropic and lusitropic responses in cells treated with the cholesterol-depleting agent methyl-β-cyclodextrin (MBCD). This was not linked to modulation of L-type Ca2+ current, but instead to a discrete PKA-mediated phosphorylation of phospholamban at Ser16. Application of a cell-permeable inhibitor of caveolin-3 scaffolding interactions mimicked the effect of MBCD on phosphorylated phospholamban (pPLB) during β2-AR stimulation, consistent with MBCD acting via caveolae. Biosensor experiments revealed β2-AR mobilisation of cAMP in PKA II signalling domains of intact cells only after MBCD treatment, providing a real-time demonstration of cAMP freed from caveolar constraint. Other proteins have roles in compartmentation, so the effects of phosphodiesterase (PDE), protein phosphatase (PP) and phosphoinositide-3-kinase (PI3K) inhibitors on pPLB and contraction were compared in control and MBCD treated cells. PP inhibition alone was conspicuous in showing robust de-compartmentation of β2-AR-derived signalling in control cells and a comparatively diminutive effect after cholesterol depletion. Collating all evidence, we promote the novel concept that caveolae limit β2-AR-cAMP signalling by providing a platform that not only attenuates production of cAMP but also prevents inhibitory modulation of PPs at the sarcoplasmic reticulum. This article is part of a Special Issue entitled “Local Signaling in Myocytes”.

Effects of cholesterol depletion on compartmentalized cAMP responses in adult cardiac myocytes.

β1-Adrenergic receptors (β1ARs) and E-type prostaglandin receptors (EPRs) both produce compartmentalized cAMP responses in cardiac myocytes. The role of cholesterol-dependent lipid rafts in producing these compartmentalized responses was investigated in adult rat ventricular myocytes. β1ARs were found in lipid raft and non-lipid raft containing membrane fractions, while EPRs were only found in non-lipid raft fractions. Furthermore, β1AR activation enhanced the L-type Ca2+ current, intracellular Ca2+ transient, and myocyte shortening, while EPR activation had no effect, consistent with the idea that these functional responses are regulated by cAMP produced by receptors found in lipid raft domains. Using methyl-β-cyclodextrin to disrupt lipid rafts by depleting membrane cholesterol did not eliminate compartmentalized behavior, but it did selectively alter specific receptor-mediated responses. Cholesterol depletion enhanced the sensitivity of functional responses produced by β1ARs without having any effect on EPR activation. Changes in cAMP activity were also measured in intact cells using two different FRET-based biosensors: a type II PKA-based probe to monitor cAMP in subcellular compartments that include microdomains associated with caveolar lipid rafts and a freely diffusible Epac2-based probe to monitor total cytosolic cAMP. β1AR and EPR activation elicited responses detected by both FRET probes. However, cholesterol depletion only affected β1AR responses detected by the PKA probe. These results indicate that lipid rafts alone are not sufficient to explain the difference between β1AR and EPR responses. They also suggest that β1AR regulation of myocyte contraction involves the local production of cAMP by a subpopulation of receptors associated with caveolar lipid rafts.

Hydrogen sulfide inhibits Cav3.2 T-type Ca2+ channels

The importance of H2S as a physiological signaling molecule continues to develop, and ion channels are emerging as a major family of target proteins through which H2S exerts many actions. The purpose of the present study was to investigate its effects on T‐type Ca2+ channels. Using patch‐clamp electrophysiology, we demonstrate that the H2S donor, NaHS (10 μM–1 mM) selectively inhibits Cav3.2 T‐type channels heterologously expressed in HEK293 cells, whereas Cav3.1 and Cav3.3 channels were unaffected. The sensitivity of Cav3.2 channels to H2S required the presence of the redox‐sensitive extracellular residue H191, which is also required for tonic binding of Zn2+ to this channel. Chelation of Zn2+ with N,N,N‘,N‘‐tetra‐2‐picolylethylenediamine prevented channel inhibition by H2S and also reversed H2S inhibition when applied after H2S exposure, suggesting that H2S may act via increasing the affinity of the channel for extracellular Zn2+ binding. Inhibition of native T‐type channels in 3 cell lines correlated with expression of Cav3.2 and not Cav3.1 channels. Notably, H2S also inhibited native T‐type (primarily Cav3.2) channels in sensory dorsal root ganglion neurons. Our data demonstrate a novel target for H2S regulation, the T‐type Ca2+ channel Cav3.2, and suggest that such modulation cannot account for the pronociceptive effects of this gasotransmitter.—Elies, J., Scragg, J. L., Huang, S., Dallas, M. L., Huang, D., MacDougall, D., Boyle, J. P., Gamper, N., Peers, C., Hydrogen sulfide inhibits Cav3.2 T‐type Ca2+ channels. FASEB J. 28, 5376–5387 (2014). www.fasebj.org

The Golgi apparatus is a functionally distinct Ca2+ store regulated by the PKA and Epac branches of the β1-adrenergic signaling pathway

Calcium released from the Golgi apparatus may promote receptor trafficking to the surface of cardiomyocytes. A distinct calcium store in cardiomyocytes In cardiomyocytes, contraction can be triggered by large influxes of calcium (Ca2+) into the cytoplasm from the sarcoplasmic reticulum. Yang et al. found that Ca2+ released from the Golgi apparatus had a distinct function from the Ca2+ stored in the sarcoplasmic reticulum. Instead of triggering contraction, Golgi-released Ca2+ promoted the trafficking of a receptor to the surface of cardiomyocytes. The adrenaline receptor β1-AR enhanced the release of Ca2+ from the Golgi through a process involving two pathways downstream of the second messenger cAMP. Cardiomyocytes from rats with experimentally induced heart failure showed increased release of Ca2+ from the Golgi, which was associated with decreased abundance of phosphodiesterases in the PDE3 and PDE4 families, which degrade cAMP. Thus, the Golgi apparatus releases Ca2+ in response to changes in cAMP concentrations, such as those that occur during adrenergic stimulation or heart failure. Ca2+ release from the Golgi apparatus regulates key functions of the organelle, including vesicle trafficking. We found that the Golgi apparatus was the source of prolonged Ca2+ release events that originated near the nuclei of primary cardiomyocytes. Golgi Ca2+ release was unaffected by depletion of sarcoplasmic reticulum Ca2+, and disruption of the Golgi apparatus abolished Golgi Ca2+ release without affecting sarcoplasmic reticulum function, suggesting functional and spatial independence of Golgi and sarcoplasmic reticulum Ca2+ stores. β1-Adrenoceptor stimulation triggers the production of the second messenger cAMP, which activates the Epac family of Rap guanine nucleotide exchange factors and the kinase PKA (protein kinase A). Phosphodiesterases (PDEs), including those in the PDE3 and PDE4 families, degrade cAMP. Activation of β1-adrenoceptors stimulated Golgi Ca2+ release, an effect that required activation of Epac, PKA, and the kinase CaMKII. Inhibition of PDE3s or PDE4s potentiated β1-adrenergic–induced Golgi Ca2+ release, which is consistent with compartmentalization of cAMP signaling near the Golgi apparatus. Interventions that stimulated Golgi Ca2+ release appeared to increase the trafficking of vascular endothelial growth factor receptor–1 (VEGFR-1) from the Golgi apparatus to the surface membrane of cardiomyocytes. In cardiomyocytes from rats with heart failure, decreases in the abundance of PDE3s and PDE4s were associated with increased Golgi Ca2+ release events. These data suggest that the Golgi apparatus is a focal point for β1-adrenergic–stimulated Ca2+ signaling and that the Golgi Ca2+ store functions independently from the sarcoplasmic reticulum and the global Ca2+ transients that trigger contraction in cardiomyocytes.

A novel approach to the Langendorff technique: preparation of isolated cardiomyocytes and myocardial samples from the same rat heart

•  What is the central question of this study? The isolation of cardiomyocytes and the homogenization of myocardium are commonly performed on separate rodent hearts. We asked whether it might be viable to derive cells as well as whole muscle (homogenized or cryopreserved) from one organ. •  What is the main finding and its importance? We provide examples of six diverse experiments that can be performed using cells and tissue samples generated by a modified Langendorff technique. The volume and power of resultant data are increased, and the method may be especially beneficial in models of cardiovascular disease.

Simvastatin promotes cardiac myocyte relaxation in association with phosphorylation of Troponin I

The number of people taking statins is set to increase across the globe due to recent changes in prescription guidelines. For example, half the US population over 40 is now eligible for these drugs, whether they have high serum cholesterol or not. With such development in policy comes a stronger need for understanding statins’ myriad of effects. Surprisingly little is known about possible direct actions of statins on cardiac myocytes, although claims of a direct myocardial toxicity have been made. Here, we determine the impact of simvastatin administration (40 mg/kg/day) for 2 weeks in normocholesterolemic rats on cardiac myocyte contractile function and identify an underlying mechanism. Under basal conditions, statin treatment increased the time to half (t0.5) relaxation without any effect on the magnitude of shortening, or the magnitude/kinetics of the [Ca2+]i transient. Enhanced myocyte lusitropy could be explained by a corresponding increase in phosphorylation of troponin I (TnI) at Ser23,24. Statin treatment increased expression of eNOS and Ser1177 phosphorylated eNOS, decreased expression of the NOS-inhibitory proteins caveolins 1 and 3, and increased (P = 0.06) NO metabolites, consistent with enhanced NO production. It is well-established that NO stimulates protein kinase G, one of the effectors of TnI phosphorylation at Ser23,24. Trends for parallel changes in phospho-TnI, phospho-eNOS and caveolin 1 expression were seen in atrial muscle from patients taking statins. Our data are consistent with a mechanism whereby chronic statin treatment enhances TnI phosphorylation and myocyte lusitropy through increased NO bioavailability. We see no evidence of impaired function with statin treatment; the changes we document at the level of the cardiac myocyte should facilitate diastolic filling and cardiac performance.