Lecture 22 PSC2002

Page 1: Introduction to cAMP Signalling

  • CAMP Signalling: Discussing dynamics of cAMP (cyclic adenosine monophosphate) signalling in cardiac cells related to GPCR (G-Protein Coupled Receptors).

  • Key Components:

    • GPCR

    • G Protein (G)

    • Phosphodiesterase (PDE)

    • Adenylate Cyclase (AC)

    • ATP as substrate.

Page 2: Learning Outcomes

  • Describe spatiotemporal changes in cAMP in cardiac cells due to GPCR agonists.

  • Discuss phenotypic remodelling in cardiac excitation-contraction coupling and the role of cAMP in calcium signalling.

Page 3: Recap of L21

  • Non-Uniform cAMP Levels: Changes are local and occur within microdomains.

  • Use of Signalsomes: Enhances signalling efficiency.

  • Calcium Measurement Techniques: Ca2+-sensitive fluorescent indicators used to measure intracellular calcium dynamics.

Page 4: Measuring cAMP Dynamics

  • Genetically-encoded Fluorescent Sensors:

    • Use cAMP binding domains (BD) combined with fluorescent proteins.

    • Measurement Techniques:

      1. FRET (Fluorescence Resonance Energy Transfer): Decrease in FRET indicates an increase in cAMP.

      2. Fluorescent Intensity: Direct measurement of increased fluorescence correlates with cAMP levels.

Page 5: cAMP-sensitive Fluorescent Sensors

  • Studies temporal & spatial changes in cAMP in living cells.

  • References: Massengil et al., Nat. Meth (2022) covering both temporal and spatial responses.

Page 6: Experimental Data

  • FRET Analysis: Visualizing cAMP changes through treatment with noradrenaline (NE) or IBMX (phosphodiesterase inhibitor) under controlled conditions.

Page 7: Differences in GPCR Agonist Responses

  • Comparison of GPCR Agonists:

    • Noradrenaline increases contractility.

    • PGE1 shows no effect on contractility.

  • FRET Experiments: An essential method for exploring spatiotemporal changes of cAMP induced by different agonists.

Page 10: Conclusions from FRET Experiments

  1. β-adrenergic Receptor Activation: Elevates cAMP localized to T-tubules and sarcoplasmic reticulum.

  2. Phosphorylation Events: Target key proteins involved in excitation-contraction coupling.

  3. Prostanoid Receptors: Lead to long-term alterations via transcription factors.

  4. Inhibition of PDEs: Abolishes cAMP signalling and PKA phosphorylation.

  5. Role of PDEs: Critical in regulating spatial cAMP signals and functional cardiac responses.

  6. Phenotypic Remodelling: Unique to each receptor, reliant on cAMP spatial changes and downstream signalling.

Page 11: Interplay with Calcium Signalling

  • cAMP/PKA Pathway Effects:

    • Increased contractility during β-adrenergic stimulation.

    • Enhancements in Ca2+ signalling components include:

      1. L-type calcium channels (LTCCs).

      2. Phospholamban (PLB).

      3. Ryanodine Receptors (RyR2).

  • Cardiac Function Enhancement:

    • Larger Ca2+ signals, faster relaxation (lusitropy), and improved Ca2+ store reloading.

Page 12: LTCC Activity Enhanced by PKA

  • Phenotypic Remodelling through PKA: Involves activation and optimization of LTCC activity through voltage variation.

Page 13: AKAP Requirement for LTCC Activity

  • Role of AKAP: AKAP-79 identified as pivotal for phosphorylation of LTCC by PKA, highlighting mechanisms of LTCC localization and activity modulation.

Page 14: Mechanism of LTCC Activity Increase

  • Increased Open State Probability (Po): PKA phosphorylation allows LTCCs to remain open longer.

  • Recruitment of LTCCs: Increases their number on the sarcolemma.

Page 15: Beta-Adrenergic Stimulation and LTCC Clustering

  • Exploration of LTCC Superclusters: GFP tagging LTCCs showed their clustering post-beta-adrenergic stimulation.

  • Receptor Interaction Dependency: PKA-induced phosphorylation leads to clustering via C-terminal domain interactions.

Page 16: Effects on SERCA Pump

  • Impact on SERCA: PKA phosphorylation of Phospholamban (PLB) lifts its inhibition on SERCA, enhancing Ca2+ uptake during diastole.

Page 17: Conclusion and Interactions

  • cAMP/PKA Signalling: Specifically localized, effectively enhancing contraction.

  • Interconnected Role of Calcium Signalling: Maximizes cardiac contractile responses.

Page 18 & 19: Disruptions in Cystic Fibrosis (CF)

  • Investigations into cAMP Signalling in CF cells: Spatial cAMP signalling challenges in CF airway cells discussed, referencing FRET studies.

Page 1: Introduction to cAMP Signalling

cAMP Signalling: This section discusses the intricate dynamics of cyclic adenosine monophosphate (cAMP) signalling within cardiac cells, specifically focusing on its interaction with G-Protein Coupled Receptors (GPCRs). cAMP serves as a secondary messenger that transduces extracellular signals into intracellular responses.

Key Components:

  • GPCR: A large family of receptors that sense molecules outside the cell and activate internal signal transduction pathways, influencing numerous physiological processes.

  • G Protein (G): These proteins relay signals from GPCRs to various intracellular targets, playing a crucial role in the modulation of cAMP synthesis.

  • Phosphodiesterase (PDE): Enzyme responsible for breaking down cAMP, thus regulating the signal duration and intensity within cells.

  • Adenylate Cyclase (AC): An enzyme that converts ATP to cAMP in response to GPCR activation, serving as a pivotal step in cAMP signalling.

  • ATP as substrate: Provides the necessary energy and phosphate group for cAMP production, underscoring the importance of cellular energy status in signalling pathways.

Page 2: Learning Outcomes

  1. Describe spatiotemporal changes in cAMP in cardiac cells: Understand how different GPCR agonists induce localized and temporally specific increases in cAMP levels, impacting cardiac function.

  2. Discuss phenotypic remodelling: Analyze how alterations in cAMP signals can lead to significant changes in cardiac excitation-contraction coupling, emphasizing the role of cAMP in calcium handling within cardiac myocytes.

Page 3: Recap of L21

  • Non-Uniform cAMP Levels: It is critical to recognize that cAMP changes are not homogeneous but localized, creating microdomains that can affect localized cellular functions.

  • Use of Signalsomes: Signalsomes aid in enhancing the efficiency of signalling processes by clustering relevant signalling molecules in specific sub-cellular locations.

  • Calcium Measurement Techniques: Advanced techniques utilizing Ca2+-sensitive fluorescent indicators are pivotal for real-time measurement of intracellular calcium dynamics, allowing researchers to study the intertwined roles of cAMP and calcium in cardiac cells.

Page 4: Measuring cAMP Dynamics

  • Genetically-encoded Fluorescent Sensors: These tools are engineered to use cAMP binding domains (BD) fused with fluorescent proteins to provide insights into cAMP levels.

  • Measurement Techniques:

    • FRET (Fluorescence Resonance Energy Transfer): This technique reveals decreases in FRET efficiency as an indication of cAMP elevation, permitting visualization of dynamic changes within live cells.

    • Fluorescent Intensity: Directly measures increased fluorescence, providing a quantitative assessment of cAMP levels over time.

Page 5: cAMP-sensitive Fluorescent Sensors

These sensors enable the study of both temporal and spatial variations in cAMP levels in living cells, benefitting research in cardiac physiology and pathophysiology.

Page 6: Experimental Data

  • FRET Analysis: This method visualizes cAMP changes following treatments with noradrenaline (NE) or the phosphodiesterase inhibitor IBMX, under controlled experimental conditions to ensure reproducibility and accuracy of results.

Page 7: Differences in GPCR Agonist Responses

  • Comparison of GPCR Agonists: Investigate the differential effects of agonists such as noradrenaline, which markedly increases cardiac contractility, versus PGE1, which exhibits no significant impact, highlighting the specificity of receptor-mediated responses.

  • FRET Experiments: This essential technique for delineating spatiotemporal cAMP changes is crucial for understanding how different agonists modulate cardiac signalling pathways.

Page 10: Conclusions from FRET Experiments

  • β-adrenergic Receptor Activation: This activation significantly elevates cAMP levels localized specifically to T-tubules and sarcoplasmic reticulum, regions critical for cardiac contraction.

  • Phosphorylation Events: Key phosphorylation of proteins involved in excitation-contraction coupling, which ultimately leads to enhanced cardiac contractility.

  • Prostanoid Receptors: Activating these receptors can lead to long-term transcriptional changes affecting cardiac function.

  • Inhibition of PDEs: This mechanism abolishes cAMP signalling and PKA (Protein Kinase A) phosphorylation, illustrating the importance of PDE in regulating these pathways.

  • Role of PDEs: PDEs are essential in controlling spatial cAMP signals and subsequent functional responses in the heart, influencing both acute and chronic cardiac adaptations.

  • Phenotypic Remodelling: Unique alterations in cardiac function are driven by distinct cAMP signalling pathways, dependent on receptor subtype and cellular context.

Page 11: Interplay with Calcium Signalling

  • cAMP/PKA Pathway Effects: Enhancing cardiac contractility during β-adrenergic stimulation illustrates the functional consequences of cAMP signalling.

  • Enhancements in Ca2+ signalling components:

    • L-type calcium channels (LTCCs): Their activity is pivotal for excitation-contraction coupling.

    • Phospholamban (PLB): Regulation of LTCC activity is crucial for calcium homeostasis.

    • Ryanodine Receptors (RyR2): Mediate calcium release from the sarcoplasmic reticulum, integral to cardiac contraction.

  • Cardiac Function Enhancement: Resulting in larger Ca2+ signals, accelerated relaxation (lusitropy), and improved reloading of calcium stores during diastole.

Page 12: LTCC Activity Enhanced by PKA

  • Phenotypic Remodelling through PKA: This process includes activation and optimization of LTCC activity, leading to improved cardiac function through voltage-dependent modulation.

Page 13: AKAP Requirement for LTCC Activity

  • Role of AKAP: A-kinase anchoring protein (AKAP-79) is essential for the targeted phosphorylation of LTCC by PKA, providing insights into the mechanisms that localize LTCC activity in cardiac cells.

Page 14: Mechanism of LTCC Activity Increase

  • Increased Open State Probability (Po): Enhancing the likelihood of LTCCs being in the open state due to PKA phosphorylation, which increases calcium influx during action potentials.

  • Recruitment of LTCCs: Increased density of LTCCs on the sarcolemma enhances overall calcium entry and, consequently, cardiac contractility.

Page 15: Beta-Adrenergic Stimulation and LTCC Clustering

  • Exploration of LTCC Superclusters: Utilizing GFP tagging, researchers observe the clustering of LTCCs following beta-adrenergic stimulation, indicating a dynamic structural change in response to signaling.

  • Receptor Interaction Dependency: The phosphorylation induced by PKA plays a critical role in this clustering mechanism, relying on interactions within the C-terminal domain of the LTCC.

Page 16: Effects on SERCA Pump

  • Impact on SERCA: PKA-mediated phosphorylation of phospholamban (PLB) relieves its inhibition on the SERCA pump, leading to enhanced calcium uptake during diastolic relaxation, which is vital for effective cardiac function.

Page 17: Conclusion and Interactions

  • cAMP/PKA Signalling: This signalling is characterized by its specific localization, which translates to efficient enhancement of cardiac contraction and performance.

  • Interconnected Role of Calcium Signalling: The interplay between cAMP and calcium signalling maximizes the contractile responses of the heart, essential for maintaining cardiac output and function.

Page 18 & 19: Disruptions in Cystic Fibrosis (CF)

  • Investigations into cAMP Signalling in CF cells: Highlight the spatial challenges faced in cAMP signalling within cystic fibrosis airway cells, with references to FRET studies that illustrate how abnormal cAMP dynamics affect cellular responses in pathological conditions.