Cardiovascular Disease - Part 2: Cellular Basis of Heart Dysfunction in Heart Failure

Introduction

This lecture delves into the cellular and molecular mechanisms that contribute to heart dysfunction, particularly in the context of heart failure. Building on the structural adaptations discussed previously, we will explore:

Methods for measuring heart function and their respective advantages and limitations (ECG, Ultrasound, Cardiac MRI, Pressure-Volume Catheterization).
The importance of the pressure-volume relationship and key parameters like End-Systolic Pressure-Volume Relationship (ESPVR) and End-Diastolic Pressure-Volume Relationship (EDPVR).
The impact of preload and afterload on cardiac function.
Diastolic dysfunction: its causes and consequences, focusing on passive myocardial properties.
The role of fibrosis, microtubules, and titin in determining myocardial stiffness and diastolic function.
Systolic dysfunction: focusing on active properties of the myocardium.
Regulation of contractility by the beta-adrenergic signaling pathway.
Beta1-adrenoceptor desensitization in heart failure.
Changes in calcium handling in heart failure, including SERCA activity and phospholamban regulation.
Alterations in myofilament properties, specifically Troponin I phosphorylation.

I. Measuring Heart Function

Several techniques are used to assess different aspects of heart function:

Electrocardiogram (ECG):
- Advantages: Provides information on electrical properties, conduction, heart rate, and rhythm. Low cost, simple to perform, non-invasive.
- Limitations: No direct information on contractile function or blood pressure. Requires specialist interpretation.
Ultrasound (Echocardiography):
- Advantages: Provides 2D/3D quantification of heart structure (chamber sizes, wall thickness) and contraction (stroke volume, ejection fraction, assessment of hypertrophy). Can use Doppler to indirectly measure pressure gradients and estimate systolic pressures. Moderate cost, fast, non-invasive. Can also assess vascular function.
- Limitations: Contraction measurements are dependent on preload (ventricular filling) and afterload (vascular resistance). No direct information on diastolic pressures across all phases of the cardiac cycle. Limited spatial resolution. Image quality can be limiting (e.g., in obesity).
Cardiac Magnetic Resonance Imaging (CMR):
- Advantages: Detailed 3D quantification of heart structure, contraction, and blood flow in all chambers. Contrast techniques (e.g., Late Gadolinium Enhancement - LGE; T1 mapping) can measure and characterize fibrosis.
- Limitations: Measurements of contractility are still influenced by preload and afterload. No direct pressure measurement. Expensive (e.g., ~£3k per scan), slow (1-2 hours), not typically real-time for all functional assessments.
Pressure-Volume (PV) Catheterization:
- Advantages: Gold standard for assessing load-independent contractility. Provides direct, real-time measurement of systolic and diastolic pressures and volumes in all heart chambers and major vessels throughout the entire cardiac cycle.
- Limitations: Invasive procedure (catheters inserted into heart chambers, e.g., via femoral artery/vein). Provides no direct information on heart structure (unless combined with imaging) or electrical function.
- Pressure-Volume Loops: By briefly occluding the vena cava to reduce ventricular filling (Frank-Starling mechanism), a series of PV loops at different preloads can be generated.
  - End-Systolic Pressure-Volume Relationship (ESPVR): The line connecting the end-systolic points of these loops. Its slope is a load-independent measure of ventricular contractility (inotropy). An increase in the slope indicates increased contractility.
  - End-Diastolic Pressure-Volume Relationship (EDPVR): The line connecting the end-diastolic points. It reflects the passive stiffness or compliance of the ventricle. A steeper EDPVR indicates increased stiffness and diastolic dysfunction.

II. Determinants of Cardiac Function: Preload and Afterload

Preload: The degree of stretch of the ventricular muscle fibers at the end of diastole, just before contraction. Primarily determined by end-diastolic volume (EDV).
- Increased preload (within physiological limits) increases stroke volume (Frank-Starling mechanism).
- On a PV loop, increased preload shifts the loop to the right (higher EDV).
Afterload: The resistance or load against which the ventricle must contract to eject blood. Primarily determined by aortic pressure and systemic vascular resistance.
- Increased afterload reduces stroke volume if contractility remains constant.
- On a PV loop, increased afterload results in a higher end-systolic pressure and often a larger end-systolic volume (reduced ejection).

III. Diastolic Dysfunction: Impaired Ventricular Filling

Diastolic dysfunction refers to the inability of the ventricle to relax and fill normally at low pressures.

Impact of Ventricular Filling (Preload): Ventricular filling is critical for determining stroke volume.
EDPVR and Stiffness: The End-Diastolic Pressure-Volume Relationship (EDPVR) indicates how easily the heart expands as it fills (its compliance or distensibility).
- A steeper EDPVR signifies increased ventricular stiffness and resistance to filling. This means higher pressures are required to achieve the same ventricular volume, or less volume is filled at a given pressure.
Consequences:
- Limits the return of blood from the body (venous congestion), potentially leading to fluid buildup in the lungs (pulmonary edema, causing shortness of breath) and peripheral tissues (e.g., legs, abdomen).
- Restricts stroke volume because end-diastolic volume is reduced or achieved at pathologically high pressures.
- LV Dilation can shift the EDPVR rightwards, initially accommodating larger volumes, but often associated with reduced contractility in failing hearts. Increased stiffness (dashed red line on PV loop) shifts the EDPVR upwards and leftwards compared to a normal EDPVR.

Passive Determinants of Myocardial Relaxation and Stiffness: These are structural components of the myocardium that influence its passive mechanical properties (resistance to stretch) without directly consuming ATP for their passive role.

A. Fibrosis:

Normal Myocardium: Contains a low amount of interstitial collagen (2-4%), mainly Type I and III fibrillar collagens, which form a structural scaffold.
Pathological Fibrosis: An excessive deposition of collagen by activated cardiac fibroblasts (myofibroblasts) in response to injury (e.g., MI) or chronic stress (e.g., hypertension).
- Can be diffuse (interstitial fibrosis, e.g., in hypertension) or localized (replacement fibrosis/scar, e.g., post-MI).
- Increased collagen content and covalent cross-linking of collagen fibers (e.g., by advanced glycation endproducts or increased lysyl oxidase activity) significantly increase myocardial stiffness.
- This impairs diastolic function (makes the ventricle harder to fill). Studies show a correlation between the extent of fibrosis (e.g., measured by Late Gadolinium Enhancement - LGE in CMR) and worse diastolic function parameters.

B. Microtubules:

Cytoskeletal proteins formed by the polymerization of α-tubulin and β-tubulin dimers.
They are dynamic structures that can assemble and disassemble. Their stability is influenced by post-translational modifications, such as the detyrosination of α-tubulin (removal of a C-terminal tyrosine residue), which makes microtubules more stable and long-lasting.
Role in Heart Failure:
- Microtubule density and the proportion of detyrosinated (more stable) microtubules increase in failing hearts.
- This increased and more stable microtubule network contributes to increased intracellular viscoelasticity (stiffness) and resistance to cardiomyocyte stretch during diastole.
- Studies on cardiomyocytes isolated from failing hearts show increased passive stiffness and stress relaxation compared to normal cardiomyocytes, which can be partly attributed to alterations in the microtubule network.

C. Titin:

The largest known protein (~3.5 MDa), spanning half a sarcomere from the Z-disk to the M-line.
Acts as a bidirectional molecular spring within the cardiomyocyte:
- Resists excessive stretch of the sarcomere during diastole (contributes to passive stiffness).
- Helps restore sarcomere length during relaxation after contraction (restoring force).
Structure: Contains multiple domains, including immunoglobulin (Ig)-like domains, Fibronectin type III (FnIII)-like domains, and a PEVK (Pro-Glu-Val-Lys rich) segment, which are responsible for its elastic properties. Also has a pseudokinase domain.
Isoforms and Modifications:
- Cardiac muscle expresses two main titin isoforms arising from alternative splicing of the single TTN gene:
  - N2B isoform: Shorter, stiffer spring region.
  - N2BA isoform: Longer, more compliant (less stiff) spring region due to the inclusion of additional elastic segments.
- The ratio of N2BA to N2B isoforms can change in heart disease, influencing overall myocardial stiffness. For example, an increase in the N2B:N2BA ratio makes the myocardium stiffer.
- Post-Translational Modifications (PTMs): Titin's compliance is dynamically regulated by PTMs, especially phosphorylation, within its spring elements (e.g., N2B-Us unique sequence, PEVK segment).
  - Phosphorylation by kinases like PKA (Protein Kinase A), PKG (Protein Kinase G), ERK2, and CaMKIIδ can decrease titin-based stiffness (make it more compliant).
  - Phosphorylation by PKCα can increase titin-based stiffness.
- In heart failure, alterations in titin isoform expression and phosphorylation status (e.g., hypophosphorylation at certain PKA/PKG sites, or hyperphosphorylation by PKCα) can contribute to increased diastolic stiffness.

Summary of Passive Properties: Extracellular (fibrosis), cytoskeletal (microtubules), and within-sarcomere (titin) elements all influence the passive stretch and stiffness of the myocardium. Dysregulation of these components in heart disease leads to increased resistance to filling (diastolic dysfunction), which negatively impacts stroke volume and can cause fluid accumulation. Pressure-volume loops are ideal for measuring these load-independent diastolic properties (EDPVR).

IV. Regulation of Contractility (Active Properties) by Beta-Adrenergic Signaling

Active properties of the myocardium relate to processes that consume ATP and directly influence the rate and force of actin-myosin cross-bridge cycling. Key determinants include cytosolic Ca2+ handling and myofilament Ca2+ sensitivity.

A. Sympathetic Nervous System and Beta-Adrenergic Signaling:

The heart is innervated by sympathetic nerves that release noradrenaline (norepinephrine), and it also responds to circulating adrenaline (epinephrine) from the adrenal medulla.
These catecholamines bind primarily to β1-adrenergic receptors (β1-AR) on cardiomyocytes, which are G-protein coupled receptors (GPCRs).
Function: This system provides a rapid response to increase cardiac output during stress ("fight or flight"). It has positive:
- Chronotropic effects: Increases heart rate (acting on the sinoatrial node).
- Inotropic effects: Increases force of contraction.
- Lusitropic effects: Increases rate of relaxation.
- Can also influence protein synthesis (long-term trophic effects).
Signaling Cascade:
1. Epinephrine/Norepinephrine binds to β1-AR.
2. β1-AR couples to a stimulatory G protein (Gs).
3. Activated Gsα stimulates adenylyl cyclase (AC).
4. AC converts ATP to cyclic AMP (cAMP), a second messenger.
5. cAMP activates Protein Kinase A (PKA), a serine/threonine-specific kinase.
6. PKA phosphorylates various target proteins in the cardiomyocyte, modifying their function to enhance contractility and relaxation. Key PKA targets include:
  - L-type Ca2+ channels (ICa): Phosphorylation increases Ca2+ influx during the action potential.
  - Ryanodine Receptors (RyR) on the sarcoplasmic reticulum (SR): Phosphorylation can increase SR Ca2+ release.
  - Phospholamban (PLB): (See below).
  - Troponin I (TnI): (See below).

B. Beta1-Adrenoceptor Desensitization in Heart Failure:

In chronic heart failure (e.g., following MI, chronic hypertension), the heart often becomes reliant on increased sympathetic drive (elevated catecholamines) to maintain cardiac output.
However, chronic β1-AR activation leads to receptor desensitization and downregulation:
- The β1-AR itself becomes phosphorylated by G protein-coupled receptor kinases (GRKs, e.g., GRK2).
- Phosphorylated receptors are bound by β-arrestins, which promote receptor internalization (removal from the cell surface) and degradation, and also uncouple the receptor from Gs.
Consequences in Heart Failure:
- Reduced expression of β1-ARs on the cell surface.
- Impaired coupling of β1-ARs to adenylyl cyclase (e.g., due to increased expression/activity of inhibitory G proteins like Gi).
- This leads to a reduced β-adrenergic responsiveness (blunted inotropic, chronotropic, and lusitropic response to catecholamines). The heart becomes less able to increase its rate and force of contraction, and to relax quickly, especially during exercise.
- Studies show significantly lower β1-AR density in failing human hearts (e.g., from patients with dilated cardiomyopathy - DCM, or ischemic cardiomyopathy - ICM) compared to non-failing (NF) hearts.

V. Active Determinants of Relaxation and Contractility: Calcium Handling and Myofilaments

Changes in the expression and post-translational modification of proteins involved in Ca2+ handling and myofilament function are central to impaired contractility and relaxation in heart failure.

A. Excitation-Contraction Coupling (Normal Heart):

An action potential (AP) depolarizes the sarcolemma and T-tubules.
This opens L-type Ca2+ channels (ICa), leading to Ca2+ influx.
This trigger Ca2+ induces a larger release of Ca2+ from the sarcoplasmic reticulum (SR) via Ryanodine Receptors (RyRs) – this is Ca2+-induced Ca2+ release (CICR).
The rise in cytosolic [Ca2+] allows Ca2+ to bind to Troponin C on the myofilaments, initiating actin-myosin cross-bridge cycling and contraction.
Relaxation occurs when cytosolic [Ca2+] is lowered by:
- Reuptake of Ca2+ into the SR by SERCA2a (Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPase) (major pathway in humans, ~70% of Ca2+ removal).
- Extrusion of Ca2+ out of the cell by the Na+/Ca2+ exchanger (NCX) and the plasma membrane Ca2+-ATPase (PMCA).

B. Altered Ca2+ Reuptake into SR in Heart Failure:

The rate of Ca2+ transient decline (and thus relaxation) must occur efficiently for proper diastolic filling, especially at faster heart rates.
Reduced SERCA2a Activity: A common finding in heart failure.
- Leads to slower Ca2+ reuptake into the SR.
- This results in slower relaxation, elevated diastolic [Ca2+], and reduced SR Ca2+ load (less Ca2+ available for subsequent release, impairing contractility).
- Ca2+ handling worsens at faster heart rates (negative force-frequency relationship).
- Studies show decreased SERCA2a protein expression in end-stage failing human hearts (e.g., DCM) compared to non-failing hearts.
Phospholamban (PLB) Regulation:
- PLB is a small protein associated with SERCA2a in the SR membrane.
- In its unphosphorylated state, PLB inhibits SERCA2a activity (acts as a brake).
- Phosphorylation of PLB (by PKA at Serine-16 or by CaMKII at Threonine-17, typically during β-adrenergic stimulation) relieves this inhibition, increasing SERCA2a activity and speeding Ca2+ uptake and relaxation.
- In Heart Failure:
  - Total PLB expression may not change significantly, leading to an increased inhibitory ratio of PLB:SERCA2a if SERCA2a expression is reduced.
  - Hypophosphorylation of PLB (reduced phosphorylation at Ser-16 and Thr-17) is often observed due to impaired β-adrenergic signaling (less PKA activity) or increased phosphatase activity.
  - This results in persistent SERCA2a inhibition, contributing to slowed Ca2+ uptake, impaired relaxation, and a blunted lusitropic (relaxation-enhancing) and inotropic (contraction-enhancing) response to β-adrenergic stimulation.
  - Studies using mutant non-phosphorylatable PLB (e.g., PLB-S16A where Serine-16 is mutated to Alanine) show a diminished lusitropic and inotropic response to β-AR stimulation (e.g., by isoproterenol), confirming the importance of PLB phosphorylation.

C. Myofilament Regulation and Troponin I Phosphorylation:

The troponin complex (Troponin T - TnT, Troponin I - TnI, Troponin C - TnC) is attached to tropomyosin on the thin (actin) filaments.
- In the absence of Ca2+, TnI binds to actin, and tropomyosin blocks myosin-binding sites on actin.
- When Ca2+ binds to TnC, it causes a conformational change in the troponin complex, which moves tropomyosin deeper into the actin groove, exposing myosin-binding sites and allowing cross-bridge formation.
Troponin I (TnI) Phosphorylation:
- Cardiac TnI has PKA phosphorylation sites at Serine-23 and Serine-24.
- Phosphorylation of TnI by PKA (during β-adrenergic stimulation) decreases the Ca2+ sensitivity of the myofilaments.
- This means that Ca2+ dissociates more rapidly from TnC at any given cytosolic [Ca2+], promoting faster relaxation (lusitropic effect). The force-pCa curve shifts to the right (higher Ca2+ needed for half-maximal force).
- In Heart Failure:
  - Reduced TnI Ser23/24 phosphorylation is often observed, again due to impaired β-adrenergic/PKA signaling.
  - This contributes to slowed relaxation and a blunted lusitropic response to β-adrenergic stimulation.
  - The myofilaments remain more sensitive to Ca2+, hindering rapid Ca2+ dissociation.

Summary of Cellular Dysfunction in Heart Failure

Heart failure involves complex alterations at the cellular level affecting both passive (diastolic) and active (systolic and diastolic) properties of the myocardium.

Diastolic Dysfunction: Increased myocardial stiffness due to fibrosis, alterations in the microtubule network, and changes in titin isoform expression and phosphorylation leads to impaired ventricular filling.
Systolic and Active Diastolic Dysfunction:
- Impaired β-adrenergic signaling: Receptor desensitization and downregulation reduce the heart's ability to respond to stress.
- Altered Ca2+ handling: Reduced SERCA2a activity and hypophosphorylation of phospholamban lead to slower Ca2+ reuptake into the SR, impairing relaxation and reducing SR Ca2+ content for subsequent contractions.
- Altered myofilament Ca2+ sensitivity: Reduced phosphorylation of Troponin I increases myofilament Ca2+ sensitivity, further slowing relaxation.

These cellular changes collectively contribute to the reduced cardiac output, impaired filling, and poor exercise tolerance characteristic of heart failure.