This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.
The heart functions mainly to continuously circulate oxygen and metabolite substrates by propelling blood to the lungs and body. Its intrinsic ability to adapt to differing loads and neuro-humoral conditions during the cardiac cycle enables tissues to be supplied with oxygen and nutrients that are vital for sustaining life. The heart is divided into four chambers which work simultaneously to regulate the normal sequential systolic and diastolic stages, namely the two superior atria and two inferior ventricles. During the diastolic stage, atrial and ventricular myocytes reside in a relaxed state to allow the subsequent refilling of the ventricles with blood. The heart spends approximately two-thirds of the time in this stage to allow the myocardium to return to an unstressed length and force.
The cardiac cycle is initiated by the sino-atrial (SAN) node, which is positioned in the wall of the right atrium and functions as the primary pacemaker of the heart. This group of specialised non-contractile cardiac myocytes naturally discharge at a high rate, causing it to over ride the action potentials produced by other cells in the heart. From the SAN node, action potentials spread over the atria, causing it to subsequently contract and propel blood to the ventricles. The depolarization wave generated is then swept across the heart to the atrio-ventricular (AV) node which filters and transmits electrical discharge to the ventricle via purkinje fibres. Cellular depolarisation plays a major part of ventricular ejection during the systolic stage, causing a large volume of blood to be forcefully ejected out of the heart to supply blood to the whole body.
The sympathetic nervous system is stimulated by effectors, i.e. acetylcholine and isoprenaline, which act on cardiac myocytes via both alpha and beta adrenergic receptors. Beta-1 and Beta-2 adrenergic receptors mediate positive inotropic responses, whereby Beta-1 adrenergic receptor is the predominant subtype in cardiac myocytes. In contrast, Beta-3 adrenergic receptor activation leads to negative inotropic effects. Beta-adrenergic stimulation of G-proteins (Gs) in the heart causes adenylyl cyclase (AC) to increase production of CAMP. Subsequently, CAMP-dependent protein kinase A (PKA) phosphorylates phospholamban (PLB) to produce a net increase in Ca2+ transient amplitude by inducing uptake of Ca2+ into the SR by SERCA through ryanodine receptors (RyRs). This causes an increased inotropic response, which leads to centripetal propagation of Ca2+ signals using mechanisms that enhance the amount of Ca2+ release into the cell. The mechanism of which the pumping of the heart relies on is called the excitation-contraction coupling.
EC coupling of cardiac myocytes
The role of Ca2+ in EC coupling
Excitation-contraction coupling is mediated by Ca2+ influx. Ca2+ is a critical regulator of cardiac function because it links the depolarisation phase during EC coupling to contraction of cardiac myocytes. EC coupling is orchestrated by increase or decrease in intracellular Ca2+ concentration to produce either a positive or negative inotropic effect respectively. For the cell to be in steady state, Ca2+ uptake by the SR (internal Ca2+ store) must be equal to the amount of Ca2+ released. Likewise, the amount that enters by ICa through NCX must also be equal to the amount extruded. The four major transporters that are involved in regulating the level of Ca2+ available for EC coupling include: (1)SR Ca2+-ATPase, (SERCA) (2)sarcolemmal Na/Ca exchange, (3)sarcolemmal Ca2+-ATPase, and (4)mitochondrial Ca2+ uniporter.
During muscle contraction, there is an influx of Ca2+ from the external surrounding of the cell through voltage dependent Ca2+ channels. There are two classes of voltage dependent Ca2+ channels exist, namely the L- and T-type Ca2+ channels. In cardiac muscles, EC coupling is initiated mainly through L-type Ca2+ channels, therefore, attention will be focussed on them. The generation of action potential causes a small amount of Ca2+ entering the cell through L-type Ca2+-channels. This small amount of Ca2+ then causes bulk release of Ca2+ from the SR. This is termed the â€œcalcium-induced calcium releaseâ€Â (CICR). In this mechanism, elevation of intracellular Ca2+ concentration activates a cluster of RyRs in the membrane of the SR, causing them to open and release Ca2+ sparks. This is a positive feedback mechanism which all aims to increase the levels of intracellular Ca2+. An increase in cytosolic Ca2+ allows actin and myosin contractile filaments to slide past each other, thus shortening the cells and producing force to propel blood. This process begins with Ca2+ binding to the N-terminus domain of the myofilament protein, troponin C(TnC). Cardiac troponin consists of TnC, troponin I (TnI) and troponin T (TnT). Ca2 binding to TnC induces a conformational change, which increases the affinity of TnC to the C-terminus of TnI and pull it off its actin-binding site. This allows tropomycin (Tm) and TnT to roll deeper into the groove between actin monomers, enabling myosin heads to interact with actin and form cross-bridges. Following this process, the resulting Tm-TnT complex subsequently shifts further into the groove to form more cross-bridges of thick and thin filaments at neighbouring sites to further promote contraction of cardiomyocytes. The interaction between actin and myosin is critical in regulating the EC coupling process, whereby a decrease in intracellular Ca2+ terminates contraction by causing the tropomyosin complex to return to the active site of the actin filament, causing Ca2+ to dissociate from troponin to produce relaxation.
The inhibition of CICR initiated by local depletion of SR Ca2+ is essential for diastolic refilling of the heart. In diastolic stage, the decline in intracellular Ca2+ level is caused by the action of NCX and SERCA pumps to compete for binding with free Ca2+ in the cytosol. Ca2+ extrusion from the cell, which occurs mainly through NCX decreases the amount of Ca2+ available for SERCA to take up into the SR stores. However, if SERCA is inhibited and the uptake of Ca2+ were prevented, the low amount of Ca2+ available for release from the SR decreases the maximum force of myocardial contraction during the systolic phase.
The ionic current produced by NCX is reversible, with a stoichiometry of three Na+ ions to one Ca2+ ion. Under physiological conditions, NCX works mainly to extrude Ca2+ out of the cell, causing subsequent Na+ influx. However, elevated Na+ concentrations can inactivate NCX, causing a reversal in this process to cause higher Ca2+ influx into the cell. The reversal of NCX works to extrude Ca2+ in relaxation.
In addition to RyRs, cardiomyocytes express another form of intracellular Ca2+ release channel known as Inositol (1,4,5)-triphosphate (InsP3). These receptors are found mainly in the nuclear envelope of atrial cells and can trigger Ca2+ release from SR. The function of IP3R is also found in smooth muscle SR and endoplasmic reticulum in many cell types. However, the rate and extent of Ca2+ release through the InsP3 receptor within the heart is much lower compared to RyRs.
EC coupling in atria and ventricular cells
Expression of SERCA in atrial and ventricular cells
The structure and function of SERCA
The expression level of SERCA pump fluctuates to regulate cardiac contractility and is influenced by several factors such as aging and thyroid levels. In guinea pigs tested under normal physiological conditions, SERCA is the main protein involved in cardiac relaxation and accounts for approximately 70% of Ca2+ removal from the cell. Three SERCA pumps have been identified: (1) SERCA 1, (2) SERCA 2, and (3) SERCA 3. SERCA2 encodes two alternative spliced transcripts by the ATP2A gene, namely SERCA2A and SERCA2B. SERCA2A is found predominantly in the heart and plays a more vital role in Ca2+ handling during EC coupling compared to SERCA2B. It promotes relaxation of heart muscles by sequestering cytosolic Ca2+ into intracellular SR stores through active transport. SERCA2B is highly associated with IP3 gated Ca2+ stores and is found mainly in the endoplasmic reticulum of cells. The expression level of SERCA in atrial cells is two-folds higher compared to ventricular cells, enabling an increased rate of Ca2+ uptake into the SR of atrial compared to ventricular cells. This accounts for the fact that atrial cells to reside in the contracted state for a shorter period of time compared to ventricular cells.
RyRs are located on the SR and are the major cellular mediator of CICR process. It contains multiple isoforms, whereby RyR1 is primarily expressed in skeletal muscles whereas RyR2 are found in high concentrations in the myocardium. RyRs function as the SR Ca2+ release channel and a scaffolding protein which localizes numerous key regulatory proteins (e.g. calmodulin) involved in buffering intra-SR Ca2+ concentration and modulating Ca2+ to the junctional complex. Both PKA and CamKII can phosphorylate and regulate ICa and RyR by enabling SR Ca2+ uptake via phosphorylation of PLB to produce muscle relaxation. PKA dependent inotropy is due mainly to increased Ica and enhanced SR Ca2+ uptake, both of which increase fractional Ca2+ release into the cell to cause contraction of cardiac myocytes. PLB is expressed much less in atrial cells, enabling increased Ca2+ reuptake by SERCA into the SR. Sarcolipin is also involved in regulating SERCA and is significantly expressed in atrial cells. It acts by directly binding to atrial cells to inhibit SERCA2A activity by stabilizing the interaction between SERCA2A and PLB, causing a decrease in the affinity of SERCA2A for Ca2+. This subsequently decreases Ca2+ transient amplitude to reduce the time of Ca2+ decay and relaxation.
Modulation of SERCA and its role in disease
SERCA pumps act to modulate the EC coupling process by sequestering Ca2+ back into the SR by active transport to promote relaxation of cardiac muscles. This is vital to enable the cell to replenish the Ca2+ stores in preparation for subsequent contraction during the systolic stage. SERCA is modulated by the small 52-amino acid phosphoprotein, PLB. Subsequent phosphorylation of PLB increases the affinity of SERCA for Ca2+. Elevation of SR Ca2+ content causes RyR to be more sensitive to intracellular Ca2+ enabling more Ca2+ to be released by CICR. During systole, the increased sensitivity can result in a greater fractional release for a given Ica trigger. This is due to a stimulatory effect of high intra SR free Ca2+ on the opening probability of RyR. During diastole, it can result in increased SR Ca2+ leak and frequency of Ca2+ sparks, which occur through spontaneous release of Ca2+ from individual couplons. Increased sensitivity of RyR and higher SR content attributed to binding of Ca2+ to calsequestrin, which binds to triadin and RyR inside the SR in a Ca2+ sensitive manner. Activation of SERCA is reversed by dephosphorylation of PLB, causing it to elicit an inhibitory effect on SERCA by binding to the transmembrane and stalk regions of the SERCA protein, preventing Ca2+ uptake into the SR. SR Ca2+ release is controlled by a negative feedback mechanism on Ca2+ influx whereby ample SR Ca2+ release causes further Ca2+ influx to be turned off. For instance, a decrease in SERCA pump level would result in a higher PLB to SERCA ratio, favouring maximal inhibition of SERCA. This results in less Ca2+ uptake into the SR, causing the heart to be in a contracted state for a longer period. In contrast, an increase in SERCA expression would result in a higher number of SERCA pump available to cause an increased rate of Ca2+ uptake into the SR, hastening cardiac relaxation. Adaptive changes to compensate for a decrease in SERCA pump level is a reduction in PLB protein level and phosphorylation of PLB.
Thapsigargin is a plant derived sesquiterpine lactone that causes a specific inhibition of the catalytic and transport activity of SERCA. The cytosolic Ca2+ signalling transients induced by electric stimuli include a rapid rise, followed by a slower decay. This increases the amount of Ca2+ pumped out of the cell and subsequently decreases the frequency of Ca2+ uptake by SERCA by increasing the requirement for Ca2+ release by SERCA. Inhibition of SERCA in cardiac myocytes decreases the rate of SR refilling. This induces a compensatory mechanism which increases NCX activity, which favours elimination of cytosolic Ca2+ through the plasma membrane. Thus, more time is required for the cell and SR to regain Ca2+. NCX is further prevented in low physiological Na+ concentrations, causing a progressive elevation of the basal diastolic level of cytosolic Ca2+ between consecutive stimuli.
Heart failure is found to be linked with decreased levels of SERCA pump protein expression or function. The SR membrane is not fully saturated, making it possible to incorporate additional Ca2+ pumps into it. It was found that over expression of SERCA2A pump protein results in increased contractility and a faster decay of intracellular Ca2+ transient. Therefore, adenoviral mediated gene transfer to restore SERCA pump activity in cardiac myocytes has positive effects in failing hearts by enhancing the Ca2+ uptake and improving rate dependent contractility and diastolic function. SR Ca2+ overload increases the amount of Ca2+ available for release, increasing the fraction of SR Ca2+ released for a given Ica trigger. The latter effect may be due to a stimulatory effect on high intra-SR Ca2+ on RyR open probability. This may also contribute to spontaneous release of SR Ca2+ during cellular Ca2+ overload. At immediately low SR Ca2+ content, CICR fails. This helps SR reload if it becomes relatively depleted and contribute to the turn off of SR Ca2+ release during EC coupling.
The aim of this study is to measure differences in SERCA protein expression between atrial and ventricular preparations and relate this to any differences in contractile function. Another aim is to determine the effects of SERCA inhibitor, Thapsigargine on contraction of cardiomyocytes.