© Borgis - Postępy Nauk Medycznych 7/2014, s. 487-495
*Urszula Mackiewicz, Joanna Kołodziejczyk, Bohdan Lewartowski
Komórkowe mechanizmy zaburzeń rozkurczu w niewydolności serca
Cellular mechanisms of diastolic dysfunction in the heart failure
Department of Clinical Physiology, Medical Center of Postgraduate Education, Warszawa
Head of Department: prof. Andrzej Beręsewicz, MD, PhD
Rozkurcz mięśnia sercowego jest złożonym kilkuetapowym procesem. W jego przebiegu wyróżniamy rozkurcz czynny i rozkurcz bierny. Rozkurcz czynny obejmuje usuwanie jonów Ca2+ z cytoplazmy i odłączenie się jonów Ca2+ od aparatu kurczliwego, po którym następuje separacja białek kurczliwych – miozyny i aktyny. Szybkość usuwania jonów Ca2+ z cytoplazmy zależy od aktywności i ekspresji ATP-azy wapniowej siateczki sarkoplazmatycznej oraz zlokalizowanego w błonie komórkowej wymiennika Na+/Ca2+. Z kolei odłączenie Ca2+ od aparatu kurczliwego zależy od wrażliwości troponiny C na jony Ca2+, która jest regulowana przez stopień ufosforylowania troponiny I. Rozkurcz bierny obejmuje przywrócenie długości spoczynkowej kardiomiocytów oraz ich spoczynkowe rozciąganie kardiomiocytów przez napływającą do serca krew. O sprawności tego etapu decydują sztywność białka cytoszkieletu – titiny, zależna od fosforylacji jej sprężystych domen, oraz skład kolagenowy macierzy zewnątrzkomórkowej oplatającej kardiomiocyty. Szczegółowe badania na poziomie komórkowym pokazują, że każdy z wymienionych etapów rozkurczu może być zaburzony w niewydolności serca. Dotyczy to przede wszystkim chorych z rozkurczową postacią niewydolności serca, u których frakcja wyrzucania lewej komory jest zachowana, a objawy są wynikiem zaburzeń rozkurczu. Skuteczna terapia tych pacjentów wymaga znalezienia metod korekty zaburzonych etapów rozkurczu.
The relaxation of cardiac muscle is the complicated process comprising two stages: active and passive. Active part of relaxation includes ATP-dependent Ca2+ removal from the cytoplasm and dissociation of Ca2+ from contractile apparatus followed by separation of contractile proteins – myosin and actin. Ca2+ removal from the cytoplasm depends on activity and expression of the two main Ca2+ transporters: sarcoplasmic reticulum Ca2+-ATPase and located in extracellular membrane Na+/Ca2+ exchanger. The rate of Ca2+ dissociation from the contractile apparatus is determined by Ca2+ sensitivity of troponin C, which, in turn, depends on the phosphorylation level of troponin I. Passive stage of relaxation process comprises the restoration of cardiomyocyte resting length and subsequent cardiomyocyte stretching through blood flowing into the heart. These processes are determined by the stiffness of titin, the important cytoskeletal protein, as well as by the collagen composition of the extracellular matrix. The titin stiffness is regulated by the phosphorylation level of its elastic domains. Detailed investigations on the cardiomyocyte and the tissue levels reveled disturbances at the each stage of relaxation process in the heart failure patients. It is especially truth for patients with preserved ejection fraction and the diastolic dysfunction as a dominant contributor to symptoms. The effective therapy of these patients requires improvement of diastolic function throughout targeting disturbed stages of the relaxation process.
Heart failure (HF) in the developed countries is diagnosed in 0.2% of population between 35 and 64 years of age. The morbidity increases with age and in the group of people over 80 exceeds 10%. The most frequent cause of HF (about 70% of cases) is ischemic heart disease, including myocardial infarction and hypertension (1-3).
HF and its symptoms, such as dyspnea, exercise intolerance and edema, were for many years connected to impaired systolic function of the left ventricle (LV) and inadequate organ perfusion. Lately, however, it has been highlighted that almost always systolic dysfunction is accompanied by diastolic disturbances. Moreover, in over 50% of patients with symptomatic HF there are only diastolic dysfunction and ejection fraction (EF) is preserved or slightly lowered (EF > 45-50%). The research showed that diastolic dysfunction is more correlated with severity of HF symptoms and patients’ hospitalization than systolic dysfunction. Isolated diastolic dysfunction in HF occur more often in older people, in patients with hypertension, obese patients and patients with type II diabetes (4-6).
Apart from intensive therapy based on multicenter studies long term prognosis in patient with HF is poor (over 50% of patient die in 4 years) (7). Ineffectiveness of treatment concerns, first of all, patients with diastolic dysfunction and preserved EF. In the last few decades survival in this group of patients did not improve (8).
One of the causes of such situation may be the fact that practically in all multicenter trials, testing effectiveness of HF therapy, patients with isolated diastolic dysfunction (FW > 45-50%) were not included in the studies.
Only a few trials were planned for patients with isolated diastolic dysfunction. They revealed that in patients with preserved EF ACE inhibitors, beta-blockers and aldosterone receptor antagonist – spironolacton did not reduce the mortality and HF hospitalization (9-13). On the other hand, statins significantly reduced mortality in patients with HF and preserved EF and did not in patients with lowered EF (14, 15). The only study which showed that the tested intervention was equally effective in patient with preserved or only mildly decreased EF and with significantly lowered EF was the study with third generation beta-blocker – nebivolol (16). The results of the above mentioned studies suggest that treatment bringing benefits to patients with lowered EF is not effective in patient with isolated diastolic dysfunction and that the pathomechanism of these two HF types is different.
The amount of patients with HF will increase due to lengthening of life time and higher survival of patients with myocardial infarction. Due to community ageing and obesity epidemic there will probably be higher percentage of patients with isolated diastolic dysfunction. Finding new effective methods of treatment aimed to improve diastolic function of LV requires deep understanding of the pathomechanism of diastolic disturbances in HF.
During diastole cardiac muscle relax. Cardiac muscle relaxation is a complex multistage process. In its course there is an active and passive component. The active relaxation is an energy consuming process comprising ATP – dependent elimination of Ca2+ from cytoplasm, and Ca2+ disconnecting from the contractile apparatus which results disconnecting between the main contractile proteins – myosin and actin. Passive relaxation involves restitution of resting sarcomere length thanks to elastic recoil generated by cytoskeleton protein titin and resting stretching of cardiomyocytes by blood flowing into the heart. The efficiency of this phase is determined by susceptibility to stretch of the titin and extracellular matrix surrounding cardiomyocytes (fig. 1) (17). Detailed research on the cellular level of heart muscle show that each of the mentioned stages of relaxation process can be disturbed in HF. Modern therapy of patients with HF should be directed to improvement of both relaxation stages.
Fig. 1. Stages of cardiac muscle relaxation.
SERCA – sarco-endoplasmic reticulum calcium ATPase; NCX – Na+/Ca2+ exchanger; ECM – extracellular matrix
DISTURBANCES OF ACTIVE RELAXATION PROCESS IN HEART FAILURE
Intracellular Ca2+ handling
The first stage of relaxation process, called active diastole, begins with Ca2+ ions elimination from cytoplasm. Before contraction there is an increase in the concentration of Ca2+ ions in cytoplasm (from about 0.1 μM to 1 μM). This increase is an effect of Ca2+ influx from the extracellular space through voltage activated L type calcium channels. This influx opens calcium channels of the sarcoplasmatic reticulum (SR), also called ryanodine receptors (RyRs). This phenomenon is called calcium induced calcium release (fig. 2). When Ca2+ concentration in cytoplasm increases over 1 μM contractile apparatus protein, troponin C, binds Ca2+ ions. After binding Ca2+ troponin C changes it’s conformation which enables interaction between main contractile proteins: myosin and actin, slipping of actin filaments between myosin ones and as a result cell shortening (contraction) (fig. 2).
Fig. 2. Cellular mechanisms of cardiac muscle relaxation. After contraction Ca2+ ions are transported to sarcoplasmatic reticulum (SR) by calcium ATPase (SERCA) regulated by fosfolamban (F) and removed to extracellular space by Na+/Ca2+ exchanger (NCX) and membrane ATPase (PMCA) (1). As a result of the decrease of Ca2+ ions concentration in cytoplasm they are released from troponin C (2) which leads to disconnecting of constrictile proteins – actin and myosin. Energy collected in elastic domains (PEVK, N2B, N2BA) of titin molecule generates elastic recoil, which leads to resting sarcomere length restoration (3). Sarcomere stretching over its resting length depends on titin susceptibility to stretch (3) as well as the expression of collagen I and III in the extracellular matrix (4).
Cardiomyocyte relaxation requires Ca2+ removing from cytoplasm. It consecutively enables disconnecting of Ca2+ from troponin C, disconnecting actin and myosin and relaxation. Three ion transporters are responsible for removal of Ca2+ from the cytoplasm after contraction (1) located in SR membranes, sarco-endoplasmatic reticulum calcium ATPase (SERCA), which transports Ca2+ ions to SR, and (2) two proteins located in the cell membrane: Na+/Ca2+ exchanger (NCX) and plasma membrane calcium ATPase (PMCA), which remove Ca2+ ions from the cell (fig. 2) (18).
In human cardiomyocytes SERCA is responsible for removal of about 70-80% of Ca2+ ions from the cytoplasm. That makes it a protein which determines the rate of active stage of relaxation process at the highest degree. Transport of Ca2+ ions to SR is energy consuming. SERCA uses about 15% of cardiomyocyte ATP. SERCA is inhibited by the protein localized in SR membranes – phospholamban. Phospholamban phosphorylation decrease SERCA inhibition. Phospholamban is phosphorylated by two kinases: protein kinase A (PKA) activated by stimulation of β-1 adrenergic receptors and by Ca2+ and calmodulin dependent kinase (CAMKII), activated by increase in the heart rate (which means also under catecholamine stimulation). In turn phosphatases PP1, PP2A and PP2B (calcineurin) are responsible for phospholamban dephosphorylation. Stimulation of Gq protein coupled receptors (such as angiotensin II or endothelin-1 receptors) or increase in wall stress in the heart muscle activates these phosphatases (19). The final level of phospholamban phosphorylation (and SERCA activity) depends on the resultant of kinases and phosphatases activity.
NCX localized in the cell membrane is the main route of Ca2+ eflux. It removes about 20% of Ca2+ ions activating contraction (PMCA is responsible for removal of about 1-4% of Ca2+ ions). NCX is not able to utilize ATP. It takes the energy required to Ca2+ transport from sodium gradient (during one Ca2+ ion removal from the cell three Na+ ions are transported into the cell) (18). All factors leading to lowering transmembrane sodium gradient (such as inhibition of Na+/K+ pump by digitalis glycosides or ion imbalances like hypokalaemia or hypomagnesaemia inhibit NCX activity and lead to intracellular Ca2+ accumulation).
SERCA activity and at a lower degree NCX determine the rate of Ca2+ ions removal from cytoplasm and thus the rate of their disconnecting from troponin C. This last process is additionally regulated by troponin C sensitivity to Ca2+ ions, which depends on the level of troponin I phosphorylation. This subunit of troponin is phosphorylated by three kinases: by PKA (similarly as phospholamban), by activated by nitric oxide (NO) and natriuretic peptydes protein kinase G (PKG) and by protein kinase C (PKC) activated under activation of Gq protein coupled receptors troponin I phosphorylation by PKA and PKG lowers troponin C sensitivity to Ca2+ ions which makes relaxation easier. On the other hand, phosphorylation by PKC has a reverse effect (20).
Summing up, the rate of the active part of relaxation depends on the rate of Ca2+ ions removal from cytoplasm and on the rate of Ca2+ ions disconnecting from troponin C. The first process mostly depends on SERCA activity regulated by the level of phospholamban phosphorylation. The second one depends on the troponin C Ca2+ sensitivity, which is regulated by the level of troponin I phosphorylation.
Neurohumoral regulation of intracellular Ca2+ handling
Regulation of intracellular Ca2+ handling and thus cardiomyocytes contraction-relaxation cycle by the sympathetic nervous system is, beside Frank-Starling mechanism, a main mechanism adjusting heart muscle to increased work in conditions of physical and psychical stress (fight or flight response). Activation of sympathetic system causes noradrenalin release from the sympathetic nerve terminals and adrenalin from the medulla of adrenal glands. Catecholamines, by activating beta-adrenergic receptors (mainly beta-1), lead to, in turn, activation of Gs protein, adenylate cyclase synthetizing cAMP and to activation of cAMP-dependent PKA. In addition, catecholamines cause increase in the heart rate which leads to CaMKII activation (21).
Activation of PKA and CaMKII by phosphorylation of a few Ca2+ handling proteins leads to increase in contraction strength as well as the rate of cardiomyocytes relaxation. The objects of phosphorylation are: L type Ca2+ channels, RyRs, phospholamban and troponin I.
Phosphorylation of L type Ca2+ channels increases Ca2+ influx and the amount of Ca2+ ions released from SR. RyRs phosphorylation increases their sensitivity to Ca2+ and makes their opening easier. Phospholamban phosphorylation increases SERCA activity, Ca2+ ions transport to SR and the amount of Ca2+ collected in the SR. Troponin I phosphorylation decreases troponin C sensitivity to Ca2+ ions and makes their disconnecting from protein easier, which promote relaxation.
Higher Ca2+ influx, higher Ca2+ sensitivity of RyRs and higher activity of SERCA are the main elements of positive inotropic catecholamine effect. Increased SERCA activity and decrease in troponin C Ca2+ sensitivity make relaxation easier and are the main elements of positive lusitropic catecholamine effect (22).
Ca2+ handling disturbances in the heart failure
In HF, in animal models as well as in human explanted hearts, there are numerous disturbances of expression and function of proteins engaged in intracellular Ca2+ handling. The most frequent observation is the decrease in expression and activation of SERCA, excessive increase in RyRs Ca2+ sensitivity to and increase in NCX expression.
Decrease in SERCA expression in explanted hearts may even reach 50%. Moreover, there is also a decrease level of phospholamban phosphorylation and thus higher inhibition of SERCA (23, 24). Lowered level of phospholamban phosphorylation in HF is not fully understood. On the one hand, due to intensive catecholamine stimulation activation of PKA and CaMKII kinases increases, which should lead to increase in phospholamban phosphorylation. On the other hand, however, the increase in the level of angiotensin II results in AT1 receptors activation and subsequent PKC activation which is the main activator of PP1 phosphatase. Additionally, in HF (particularly in its diastolic form) activity of PP2B phosphatase (calcineurin) increases (25, 26). It seems that the resultant effect is the predominance of phosphatases effect over kinase, which leads to decrease in phospholamban phosphorylation, decrease in SERCA activity and finally lowering the rate of relaxation. In addition, the decrease in SE CA expression and activity leads to lowering of the SERCA2+ content and to the decrease in contraction amplitude.
The following element of cellular remodeling in HF is the increase of RyRs Ca2+ sensitivity. It results in spontaneous opening of channels even at the resting Ca2+ concentration (so called Ca2+-leak) (27). This phenomenon has a lot of detrimental effects. First of all, it leads to the increase in resting Ca2+ ions concentration in cytoplasm, which prevents complete disconnection of Ca2+ from troponin C and impede relaxation process. Secondly, the SR Ca2+ content decreases which results in lowering of contraction strength. Thirdly, diastolic Ca2+-leak from SR has a pro-arrhythmic effect because it activates NCX. NCX generates depolarizing current that evokes afterdepolarizations, which may induce premature beats and arrhythmias (28). Increase in Ca2+-leak is caused by excessive RyRs phosphorylation by PKA and CaMKII. In addition, this effect is intensified by disconnecting phosphatases from RyRs. This is a different situation than phospholamban, where active effect of phosphatases prevailed over kinase activity and decided about the decrease in its phosphorylation (29). These observations highlight the fact of a very complicated regulation of Ca2+-handling proteins and complexity of their disorders in HF.
The next change in Ca2+ handling observed in HF is the increase in NCX expression. In extreme cases in the explanted hearts there was an increase in the NCX expression even by 100%. It is probably a change that is aimed to compensate decrease in SERCA expression. However, this compensation is slight. The time constant of the rate of Ca2+ removal from the cytoplazm realized by NCX is about 8-10 times lower than by SERCA (30). In addition, the increase in NCX expression leads to decrease in contraction strength because Ca2+ ions transported by NCX leave the cell and do not enter SR.
The above disturbances in expression and activity of the main proteins involved in Ca2+ handling are the cause of cardiomycytes systolic as well as diastolic dysfunction in an filing heart.
The Ca2+ handling disorders in HF at least partly can be explained by the excessive catecholamine stimulation. One of the pathomechanism of HF are the disturbed sympathetic – parasympathetic balance. The activity of sympathetic system increases while parasympathetic one decreases. The level of catecholamines in the plasma of the patients with advanced HF increase even by 100% and in the heart muscle tissue even 50 times. Additionally, the increase in catecholamine level and beta-1 receptors activation in the juxtaglomerular complex cells of the kidneys leads to renin production and consequently increased production of angiotensin II and aldosterone (RAAS system activation) (31). In literature the fact that excessive catecholamine stimulation in HF, increasing the degree of RyRs phosphorylation, which leads to excessive RyRs activity and spontaneous release of Ca2+ ions from the SR is well documented (29, 32, 33). Beta-blockers lower the spontaneous Ca2+ release from the SR by lowering the degree of RyRs phosphorylation leading at the same time to the increase of SERCA expression (34). It is also proved that the increase in mRNA NCX expression correlates positively with the increase in catecholamine level in the blood of patient suffering from HF (35).
Ca2+ sensitivity of contractile apparatus in heart failure
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