Cellular Basis for Heart Failure

Normal Excitation-Contraction Coupling

Physiologic cardiac function requires the coordinated temporal and spatial activation of the heart. At a cellular level, this finely tuned process is regulated mainly by accurately synchronized Ca ²⁺ fluxes in every cardiomyocyte ( Fig. 2.1 ). When an action potential depolarizes the cell, voltage-dependent L-type Ca ²⁺ channels (LTCCs) located mainly in the transverse tubules (T-tubules) open to generate an inward Ca ²⁺ current (I _Ca ), which induces a localized increase of Ca ²⁺ in the “dyadic cleft” in close neighborhood to the Ca ²⁺ release or ryanodine receptor channels (RyR2) of the sarcoplasmic reticulum (SR). This transsarcolemmal Ca ²⁺ influx activates the RyR2 and results in so-called Ca ²⁺ -induced Ca ²⁺ release from the SR, which provides the major component of the increase in cytosolic Ca ²⁺ during systole (i.e., the “Ca ²⁺ transient”). Intracellular Ca ²⁺ concentration increases from approximately 100 nmol/L during diastole to approximately 1 μmol/L during systole and causes myofilament activation and contraction. Repolarization of the membrane potential is induced by inactivation of I _Ca and the activation of delayed rectifying K ⁺ currents. During diastole, Ca ²⁺ is removed from the cytosol via two major pathways: (1) the SR Ca ²⁺ ATPase (SERCA2a) located in the membrane of the SR, which pumps Ca ²⁺ back into the SR lumen; (2) the sarcolemmal Na ⁺ /Ca ²⁺ exchanger (NCX1), which transfers Ca ²⁺ into the extracellular space. Through these two Ca ²⁺ transport mechanisms, [Ca ²⁺ ] _i decreases to physiologic resting concentrations of approximately 100 nmol/L, allowing the cell to relax and to regain its physiologic diastolic resting cell length.

Impaired Ca ²⁺ Handling in Failing Cardiac Myocytes

Prolongation of the action potential duration, depressed force generating capacity, and slowed contraction and relaxation rates are the hallmark functional changes of the failing human heart. Impaired Ca ²⁺ handling is a key feature of the failing cardiac myocyte, with great pathophysiologic relevance for the progressive deterioration in contractile function of the failing heart. Distinct alterations in the expression levels, as well as posttranslational modifications of important cardiac Ca ²⁺ -handling proteins, causatively contribute to systolic and diastolic contractile dysfunction and to an increased propensity for cardiac arrhythmias. The posttranslational modifications that alter the function of key Ca ²⁺ -handling proteins include alterations in phosphorylation, nitrosylation, oxidation status, and sumoylation. Altered protein phosphorylation occurs secondary to changes in the activity of various kinases (e.g., cAMP-dependent protein kinase [PKA], calcium-calmodulin-dependent kinase II [CaMKII]), as well as perturbations in phosphatase (e.g., protein phosphatase 1 [PP1]) activity.

The failing cardiac myocyte has a significantly diminished amplitude of the systolic Ca ²⁺ transient as compared with nonfailing control myocytes ( Fig. 2.2 ), which is a major factor responsible for the reduced contractile amplitude of the failing cell ( systolic dysfunction, see Chapter 10 ). Failing myocytes typically also exhibit a slowed decay of the Ca ²⁺ transient during diastole, which is a major contributor to abnormal (delayed) relaxation. In addition, the normal increase in amplitude of Ca ²⁺ transient (and therefore force of contraction) that occurs with faster heart rate is blunted or even reversed in the failing heart (i.e., the normal positive force-frequency relationship [FFR] is converted to a flat or a negative FFR). It is generally accepted that a reduction in the Ca ²⁺ content of the SR is a major reason for the diminished amplitude of the systolic Ca ²⁺ transient and the abnormal FFR. A decreased SR Ca ²⁺ content has been consistently observed in myocytes isolated from failing human and animal hearts, whereas alterations in LTTC-mediated Ca ²⁺ influx appear to be less relevant. From a mechanistic point of view, a reduction in SR Ca ²⁺ content can result either from insufficient diastolic Ca ²⁺ refilling (or loading) of the SR or from an increased loss of Ca ²⁺ via the RyR2 Ca ²⁺ release channels during diastole and may also be influenced by changes in NCX activity. In fact, all three mechanisms may contribute to the reduction in SR Ca ²⁺ content and contractile phenotype of the failing myocyte ( Fig. 2.3 ).

Reduced SR Ca ²⁺ Reuptake in Heart Failure (see also Chapter 1 )

During diastole, SERCA2a pumps Ca ²⁺ into the SR lumen and provides a sufficient Ca ²⁺ content to be released during the subsequent systolic heartbeat. SERCA2a-dependent diastolic Ca ²⁺ uptake into the SR normally dominates over transsarcolemmal Ca ²⁺ extrusion via the NCX. SERCA2a is subject to regulation by the phosphoprotein phospholamban (PLB), which on phosphorylation by CaMKII (at Thr-17) and/or PKA (at Ser-16) releases its inhibitory effect on SERCA2a because of a dissociation of the PLB/SERCA2a complex. SERCA2a protein levels are reduced in failing myocardium, which is paralleled by a reduction in SERCA2a sumoylation, and results in an impairment of diastolic Ca ²⁺ reuptake into the SR. Moreover, the levels of PLB are unaltered and its phosphorylation state may be reduced, so that there is greater relative inhibition of SERCA2a by nonphosphorylated PLB, thereby aggravating the impairment of Ca ²⁺ reuptake ( see Fig. 2.3 ). The decrease in SR Ca ²⁺ content results in less Ca ²⁺ available for the subsequent systolic Ca ²⁺ transient and impairs systolic function.

Decreased SERCA2a expression may also affect diastolic function of the failing heart. If no other mechanism (such as an increased NCX activity [see later discussion]) compensates for the reduction in SERCA2a function with respect to removal of Ca ²⁺ from the cytosol during diastole, then there is diastolic cytosolic Ca ²⁺ overload. Myocytes that have increased diastolic Ca ²⁺ levels will have persistent low-level myofilament activation at a time when the myofilaments should be fully relaxed, resulting in increased diastolic force and diastolic dysfunction. This failure to relax fully during diastole impairs the filling of the heart and thereby may also worsen systolic dysfunction. Moreover, the abnormally elevated diastolic Ca ²⁺ levels may have multiple other effects, such as changes in gene transcription, cell viability, and mitochondrial function through altered activation of Ca ²⁺ -dependent kinases (e.g., CaMKII), phosphatases (e.g., calcineurin), mitochondrial enzymes, caspases, and other mechanisms.

In view of these effects on contractile function, as well as other aspects of the failing heart phenotype, restoring SERCA2a function in heart failure might represent a promising therapeutic approach. Experimental studies showed that adenoviral overexpression of SERCA2a in human cardiomyocytes can improve cardiac contractility because it restores SR Ca ²⁺ content and the systolic Ca ²⁺ transient, whereas reduced cytosolic Ca ²⁺ levels preserve diastolic function. In addition, SERCA2a overexpression was shown to improve myocardial energetics and endothelial function and to have antiarrhythmic effects. The potential clinical relevance of SERCA2a stimulation has been addressed in the CUPID trials (Calcium Up-Regulation by Percutaneous Administration of Gene Therapy in Cardiac Disease) in which patients with heart failure were treated with a single infusion of an adeno-associated viral (AAV) vector delivering SERCA2a versus placebo. Although this initial study suggested potential efficacy, the larger study CUPID2 trial failed to show efficacy in advanced heart failure.

Increased NCX Activity in the Failing Heart

The NCX is localized at the cardiomyocyte sarcolemma where, in its “forward mode,” it transfers one Ca ²⁺ ion into the extracellular space in exchange for three Na ⁺ ions using the transmembrane gradient for Na ⁺ ( see Fig. 2.1 ). This mechanism is electrogenic because it results in the net movement of one positive charge into the cytosol and can therefore depolarize the membrane potential and even have arrhythmogenic effects under conditions of spontaneous and localized rises in [Ca ²⁺ ] _i (see later discussion). NCX expression and activity are found to be increased in human and experimental heart failure, which may have complex functional effects depending on the mode of NCX activity and stage of heart failure. In the face of downregulated SERCA2a function, enhanced NCX activity competes with SERCA2a for Ca ²⁺ elimination during diastole. This may further aggravate the decrease in SR Ca ²⁺ content because less cytosolic Ca ²⁺ is available for SERCA2a-mediated SR Ca ²⁺ loading. However, the increase in NCX function can also partly protect cardiac myocytes against severe diastolic Ca ²⁺ overload and diastolic dysfunction. Indeed, an increase in NCX levels in explanted human myocardial samples was found to correlate with a preservation of diastolic function, whereas patients with diastolic dysfunction had decreased NCX levels. On the other hand, NCX activity can contribute to Ca ²⁺ overload in settings where there is intracellular Na ⁺ overload. The reason is that at high [Na ⁺ ] _i , NCX switches to a “reverse mode” and pumps out Na ⁺ in exchange for Ca ²⁺ . The increased contribution of NCX-dependent Ca ²⁺ influx, as opposed to SR Ca ²⁺ release during systole in failing myocytes, has adverse effects on mitochondrial Ca ²⁺ uptake (which relies on high Ca ²⁺ gradients), and promotes increased mitochondrial reactive oxygen species (ROS) levels because of reduced activity of Ca ²⁺ -dependent Krebs cycle dehydrogenases that normally maintain antioxidant reserves. This detrimental mechanism can become further aggravated in a ROS-dependent manner and lead to a vicious cycle of impaired cytosolic and mitochondrial Ca ²⁺ fluxes and increased oxidative stress because ROS induce further cytosolic Na ⁺ overload.

“Leaky” RyR2 Cause Diastolic SR Ca ²⁺ Loss in Heart Failure

Diastolic “leak” of Ca ²⁺ from the SR due to a pathologic increase in RyR2 open probability is an important mechanism that contributes to the lowering of SR Ca ²⁺ content in heart failure. Ca ²⁺ leaks from the SR through spontaneous and uncoordinated Ca ²⁺ release events or “Ca ²⁺ sparks.” The expression of RyR2 itself appears to be unchanged in heart failure, but its functional regulation is dramatically altered by complex posttranslational modifications. These alterations involve an increase in RyR2 phosphorylation as a result of hyperactive protein kinases, such as CaMKII and PKA, and possibly reduced RyR2 dephosphorylation. An increase in RyR2 oxidation or nitrosylation as a consequence of increased oxidative and nitrosative stress in heart failure may also be important. Although transient phosphorylation- and redox-dependent regulation of RyR2 gating may fulfill physiologic functions in healthy myocytes, in the failing myocyte the hyperphosphorylation and/or oxidation of RyR2 leads to severe diastolic SR Ca ²⁺ leakage. Furthermore, the coupling of LTCC to RyR is also impaired in heart failure because of T-tubule remodeling, such that some RyR are “orphaned” and contribute to dyssynchronous Ca ²⁺ release.

The precise mechanisms of RyR2 hyperphosphorylation and the kinases responsible for this abnormality are important to establish because they may represent therapeutic targets but are still a matter of debate. Although one laboratory reported strong evidence for a PKA-mediated dysregulation of the RyR2 in heart failure, others failed to show an increase in PKA-dependent hyperphosphorylation. There is also evidence for an involvement of CaMKII-dependent RyR2 phosphorylation in inducing SR Ca ²⁺ leak. Redox-related dysregulation of RyR2 opening in the failing heart may involve increased ROS produced by mitochondria or other sources such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidases ( see also Chapter 8 ), and is related to the oxidation of specific cysteine residues within the RyR2. Interestingly, both the protein kinases implicated in RyR2 hyperphosphorylation (i.e., CaMKII and PKA) are subject to redox activation, so that alterations in the redox milieu of failing myocytes may also exert indirect effects on RyR2. In this regard, it is interesting to note that increased ROS in heart failure also impact adversely on SERCA2a function and other aspects of Ca ²⁺ handling in the failing cell ( see Fig. 2.2 ). More recently it has been shown that an increase in calcium leak from RyR2 results in mitochondrial calcium overload, which in turn has been linked to heart failure progression.