A pictorial representation of adjacent cardiomyocytes illustrating the genes implicated in Mendelian forms of atrial fibrillation and the presumed mechanism of action of the mutations.
The majority of mutations have been identified in ion channel subunit genes and lead to either gain-of-function or loss-of-function effects. Gain-of-function potassium channel mutations have been identified in KCNQ1, KCNE2, KCNE5 and KCNJ2. KCNQ1, KCNE2 and KCNE5 encode subunits of the cardiac IKS channel while KCNJ2 encodes a subunit of the IK1 channel. A loss-of-function potassium channel gene mutation has been reported in KCNA5, which encodes a subunit of the IKur channel. Both gain-of-function and loss-of-function mutations have been identified in SCN5A, which encodes the α subunit of the cardiac sodium channel. Loss-of-function mutations have also been reported in SCN1B and SCN2B, genes encoding β subunits of the cardiac sodium channel.
Non-ion channel gene mutations have also been implicated in familial AF. These include mutations in NUP155, GJA5 and NPPA. NUP155 encodes a nucleoporin, which is a molecular component of nuclear pore complexes. GJA5 encodes connexin-40, an atrial gap junction protein which plays a role in cell-to-cell electrical coupling. The reported NPPA mutation is associated with markedly elevated levels of mutant atrial natriuretic.
Altered Subcellular Ca2+ Handling Protein Expression and Phosphorylation in Atrial Fibrillation-induced Remodeling
Schematic drawing depicting changes in function, expression, and phosphorylation levels of key Ca2+ handling proteins in human atrial fibrillation (AF), with arrows denoting direction of change. Dephosphorylation of channel subunits due to increased activities of phosphatases 1 and 2 (PP1, PP2) contributes to the reduction of L-type Ca2+ current (ICaL), which is a hallmark of AF-induced remodeling (Inset). However, locally increased phosphorylation of (PP1)-inihibitor-1 (I-1) prevents dephosphorylation of Ca2+ release channels (Ryanodine receptors, RyR2) of the sarcoplasmic reticulum (SR), resulting in their hyperphosphorylation by protein kinase A (PKA) and Ca2+/calmodulin–dependent kinase II (CaMKII) in AF. Contribution of this hyperphosphorylation to initiation and perpetuation of AF is currently debated. Inositol 1,4,5-trisphosphate type 2 receptor (IP3R) protein expression is increased in AF, although little is known regarding its functional relevance. Upregulation of Na+/Ca2+ exchanger (NCX) function and expression is a constant finding in AF and might contribute to cellular proarrhythmic mechanisms (triggered activity). The pump rate of the SR Ca2+-ATPase (Serca2a) is increased by phosphorylation of its regulatory protein phospholamban (PLB). Similar to RyR2, PLB is hyperphosphorylated in AF at PKA and CaMKII sites (Ser16 and Thr17, respectively).
Upregulation of Na+/Ca2+ exchanger (NCX) function and expression is a constant finding in AF and might contribute to cellular proarrhythmic mechanisms (triggered activity). The pump rate of the SR Ca2+-ATPase (Serca2a) is increased by phosphorylation of its regulatory protein phospholamban (PLB). Similar to RyR2, PLB is hyperphosphorylated in AF at PKA and CaMKII sites (Ser16 and Thr17, respectively). Regional alterations in Ca2+ handling protein expression and phosphorylation show the importance of differential regulation of subcellular Ca2+ handling domains in AF.
MyBP-C, myosin-binding protein C; P, phosphate group.
*changes in MyBP-C phosphorylation are controversial in the literature.
In response to a variety of stimuli, cardiac fibroblasts proliferate, differentiate, synthesize extracellular matrix (ECM) proteins, and produce cytokines like transforming growth factor-beta (TGFβ) and interleukin-6 (IL6) that in turn stimulate fibroblasts, thereby providing positive feedback that amplifies and perpetuates the fibrogenesis cascade. ECM accumulation causes fibrosis that favours the occurrence and maintenance of AF. There are two types of fibrosis, responsive and reparative, that can be caused by many common stimuli, with reparative fibrosis being particularly involved in repairing tissues after cardiomyocyte death. Fibrosis promotes AF by acting as a conductive barrier that impedes impulse propagation and/or via proarrhythmic cellular interactions between cardiomyocytes and fibroblasts. Intracellular Ca2+ signals mediated by transient receptor potential (TRP) channels, in particular TRP melastatin-related 7 (TRPM7) channels, are critical for fibroblast proliferation, differentiation, and ECM production in fibroblasts from AF patients. Ca2+ release may affect fibroblast function via the modulation of gene expression through Ca2+-dependent transcription factors (TFs). Fibroblast Ca2+ signalling may be an effective target for the prevention of fibrogenesis and could be a novel approach to AF therapy.
Expression of skeletal muscle sodium channel (Nav1.4) or connexin32 prevents reperfusion arrhythmias in murine heart
Overexpressing the acidosis-resistant connexin32 (Cx32) maintains normal conduction in myocardium exposed to a low pH environment. Intracellular acidification induced by acute myocardial ischemia can close pH-sensitive Cx43 gap junctions and contribute to conduction slowing and reentrant arrhythmias. We studied right ventricular epicardium from mice whose hearts had been injected 4 days earlier with either adenoviral Cx32 or naked adenovirus. We simulated acidosis using a low pH solution containing Na-acetate. This decreased intracellular pH (measured with pH-sensitive microelectrodes) from 7.0 to 6.4. At pH 6.4, most Cx43 gap junctions are closed, while significant numbers of Cx32 junctions should remain open and support conduction.
In representative maps of conduction times (A – D) the pacing site is marked with a cross and isochrones are drawn at 3 ms intervals. Low pH had no effects on maximum diastolic potential (MDP) (E) and maximum upstroke velocity of the action potential (Vmax) (F) in both groups. Acidosis induced a significant decrease in conduction velocity (CV) in Shams and had no effect in Cx32 (G). As a result, at low pH, CV was higher in Cx32-expressing mice. *P<0.05 vs. pH 7.4 in the same group, +P<0.05 vs. Sham at the same pH (n=8 for both groups).
Mechanisms of atrial structural changes caused by stretch occurring before and during early atrial fibrillation
A schematic representation of the calcium homeostasis and events that may take place due to calcium overload induced by AF. Depolarization of the cardiomyocyte leads to inflow of calcium into the cell via (i) L-type Ca2+-channels, (ii) reverse mode Na+/Ca2+-exchanger, and (iii) T-type Ca2+-channels. This induces calcium-induced-calcium release from the sarcoplasmic reticulum via ryanodine receptors (RyR) into the cytoplasm. Calcium, subsequently, binds to contractile elements and initiates contraction. In the diastole, calcium leaves the cytoplasm via sarcoplasmic reticulum Ca2+-ATPase (SERCA), which is regulated by phospholamban (Pln), and via the Na+/Ca2+-exchanger and plasma membrane Ca2+-ATPase.
Ions can also leave and enter the cell via stretch-activated channels (SAC). During AF calcium overload can contribute to an altered signal transduction. Activation of Ca2+-dependent proteins such as calpain, calcineurin, and calcium/calmodulin-dependent protein kinase II (CaMKII) may be increased. Activation of calpain may result in degradation of muscle proteins (myolysis). Increased calcineurin activation activates NFAT (nuclear factor of activated T-cells) by dephosphorylation and CaMKII activates myocyte enhancer factor 2 (MEF2) signalling. Both lead to altered gene expression such as increased ANP and BNP expression and hypertrophy. Calsarcin, a stretch-sensitive protein localized to the Z-disk, is an inhibitor of calcineurin. The plasma membrane calcium-ATPase fine tunes diastolic calcium levels and inhibits calcineurin via direct binding.
KATP channel-dependent metaboproteome decoded: systems approaches to heart failure prediction, diagnosis, and therapy
Forecasting cardiac outcome from a presymptomatic proteomic signature. (A) At baseline, no differences were observed in cardiac structure or function between age- and sex-matched wild-type and Kir6.2 KATP channel knockout cohorts. Left ventricular tissue was extracted for proteomic analysis by comparative 2D gel electrophoresis resolution. (B) Statistical analysis of quantified 2D gel images indicated significant differences in 9% of detected protein species, subsequently isolated and identified by tandem mass spectrometry and categorized by primary protein function, revealing a metabolism-centric theme of protein change. (C) Altered proteins served as focus proteins for network analysis, with Ingenuity Pathways Knowledge Base expanding the KATP channel-dependent changes into a broader network neighbourhood, which reinforced the metabolic focus of measured changes both by ontological function (shown) and by ontological assessment of overrepresented biological processes (not shown).34
(D) Bioinformatic interrogation of proteome changes and their expanded network, for the presence of potential adverse effects, indicated an overrepresentation of markers associated with susceptibility to cardiac disease. Subsequent experimental imposition of graded stress validated disease susceptibility, with the Kir6.2 deficient cohort exhibiting progressively deleterious structural and functional cardiac defects, ultimately decreasing survival. *P< 0.05 vs. WT counterparts; **P< 0.01 vs. WT counterparts.
Schematic illustration of progressive contractile and conductive maturation during cardiogenesis. Schematic representation of the different stages of mouse cardiac development: cardiac crescent (A) straight tube, (B) looping, (C) embryonic, (D) foetal, (E), and adult (F) heart. The progressive formation of contractile and cell-to-cell conductive elements is depicted in (A′)–(F′). Cell-to-cell wiring illustrates the progressive alignment of connexins during cardiomyocyte differentiation. Similarly, progressive development of the cardiac action potential and the ECG recordings are illustrated in (A″)–(F″) and (A′′′)–(F′′′), respectively. Cardiac action potential diversity between conductive and working myocardium is illustrated at embryonic (E8.5) stages and between nodal, atrial, and ventricular myocytes from late embryonic stages (E9.5) onwards. Note that while cardiac action potential configuration resembles that of man, yet significant differences are observed in other species. Asterisks demarcate those stages and pathways in which impaired expression and/or function has been documented on Pitx2 deficiency. FHF, first heart field; SHF, second heart field; oft, outflow; ift, inflow; pv, primitive ventricle; rv, right ventricle; ra, right atrium; lv, left ventricle; la, left atrium; avc, atrioventricular canal.
Function and regulation of phosphatase-1-inhibitor-1 (I-1) and constitutively active I-1c.
Control of protein phosphorylation/dephosphorylation events occurs through regulation of protein kinases and phosphatases. The phosphatase type 1 comprises the main activity of Ser/Thr phosphatases in the heart. Inhibitor-1 (I-1) specifically inhibits phosphatase-1. I-1 was found to be downregulated in human heart failure but hyperactive in human atrial fibrillation, implicating I-1 in the pathogenesis of heart failure and arrhythmias. (A) I-1 represents a distal element of Β-adrenoceptor (AR) signalling, which allows amplification of protein kinase A (PKA)-mediated effects on the phosphorylation state of regulatory proteins. (B) I-1 consists of 171 amino acids (aa) and an N-terminal consensus motif (KIQF) that is essential for I-1 binding to phosphatase-1. I-1 becomes activated upon phosphorylation by cAMP-dependent PKA at Thr35, resulting in a potent inhibition of phosphatase-1. In contrast, phosphorylation at Ser67 by protein kinase Cα (PKCα) attenuates its inhibitory activity towards phosphatase-1. Phosphatase-2A (PP-2A) and Ca2+-dependent phosphatase-2B (PP-2B, calcineurin) dephosphorylate I-1 at Thr35 and thus reverses its inhibitory activity on phosphatase-1. (C) Replacement of Thr35 by phosphomimetic aspartic acid (T35D) and C-terminal truncation to 65 aa yields a constitutively active form of I-1 (I-1c) that is independent of PKA, PP-2A, and PP-2B.
Disruption of fuel servicing in the myocardium: from ‘eutaxy and efficiency’ to ‘ataxy and inefficiency’
Deletion of PGC-1β leads to disruption of mitohormesis and arrhythmias in cardiomyocytes.
A tentative scheme is shown that depicts how PPAR-γ or PGC-1s stimulation and suppression modulate electrical activity in cardiomyocytes. For biological activity, PPAR-γ needs coactivation by PGC-1α/β. These coactivators are inducible and play a vital role in cellular ATP production and mitochondrial function.
Exogenous stress stimulates the expression of chemokines and adhesion molecules in the heart. These molecules promote atrial fibrosis on the one hand, and macrophage recruitment and inflammation on the other, which result in heart failure and arrhythmias substrate formation.
The suppression or deletion of PGC-1β seems to result in variable electrical instabilities and arrhythmias, especially during adrenergic stress. An imbalance of mitochondrial ATP production is noted as well, which is arrhythmogenic by itself. The stimulation of PPARγ; blocks stress-induced expression of chemokines and adhesion molecules. The recruitment of macrophages from storage sites such as splenic red pulp (red bar) is also suppressed, resulting in an improvement of atrial fibrosis and mitochondrial function. Details of signaling cascades mediated by the PPARγ-PGC-1 axis that leads to electrical instability are waiting to be elucidated.