Background Human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs) monolayers generated to date display an immature embryonic-like functional and structural phenotype that limits their utility for research and cardiac regeneration. In particular, the electrophysiological function of hPSC-CM monolayers and bioengineered constructs used to date are characterized by slow electrical impulse propagation velocity and immature action potential profiles. Methods and Results Here we have identified an optimal extracellular matrix (ECM) for significant electrophysiological and structural maturation of hPSC-CM monolayers. hPSC-CM plated in the optimal ECM combination have impulse propagation velocities ~2X faster than previously reported (43.6±7.0 cm·s−1 n=9) and have mature cardiomyocyte action potential profiles including hyperpolarized diastolic potential and rapid action potential upstroke velocity (146.5±17.7 V/s, N=5 monolayers). In addition the optimal ECM promoted hypertrophic growth of cardiomyocytes and the expression of key mature sarcolemmal (SCN5A, Kir2.1 and Connexin43) and myofilament markers (cTroponin I). The maturation process reported here relies on activation of integrin signaling pathways: neutralization of β1 integrin receptors via blocking antibodies and pharmacological blockade of focal adhesion kinase (FAK) activation prevented structural maturation. Conclusions Maturation of human stem cell derived cardiomyocyte monolayers is achieved in a one week period by plating cardiomyocytes on PDMS coverslips rather than on conventional 2D cell culture formats such as glass coverslips or plastic dishes. Activation of integrin signaling and FAK are essential for significant maturation of human cardiac monolayers.

Key Words: plakophilin-2 Ⅲ intercalated disc Ⅲ arrhythmogenic right ventricular cardiomyopathy Ⅲ cardiac desmosomes A high-resolution image of the site of end-end contact between cardiomyocytes reveals an electron-dense organization called "the intercalated disc." Its classic definition involves 3 structures: desmosomes and adherens junctions, providing mechanical coupling; and gap junctions, allowing electric/metabolic synchronization between cells. Recent studies show that other molecules, not directly involved in intercellular coupling, also reside preferentially at the intercalated disc. Among them is Na V 1.5, the major ␣ subunit of the cardiac sodium channel. 1 Here, we ask whether Na v 1.5 and the desmosomal protein plakophilin-2 (PKP2) coexist in the same molecular complex and whether loss of PKP2 expression affects (1) the amplitude and kinetics of the sodium current and (2) action potential propagation in a monolayer of cardiomyocytes. Our data demonstrate a functional crosstalk between a protein defined in the context of intercellular junctions (PKP2) and another protein that is fundamental to the electrical behavior of the single myocyte.

Abstract-Desmosomes and gap junctions are distinct structural components of the cardiac intercalated disc. Here, we asked whether the presence of plakophilin (PKP)2, a component of the desmosome, is essential for the proper function and distribution of the gap junction protein connexin (Cx)43. We used RNA silencing technology to decrease the expression of PKP2 in cardiac cells (ventricular myocytes, as well as epicardium-derived cells) obtained from neonatal rat hearts.We evaluated the content, distribution, and function of Cx43 gap junctions. Our results show that loss of PKP2 expression led to a decrease in total Cx43 content, a significant redistribution of Cx43 to the intracellular space, and a decrease in dye coupling between cells. Separate experiments showed that Cx43 and PKP2 can coexist in the same macromolecular complex. Our results support the notion of a molecular crosstalk between desmosomal and gap junction proteins is an inherited disease that presents with sustained monomorphic ventricular tachycardia and sudden cardiac death. The disease is characterized by progressive fibrofatty infiltration of the myocardium, most prominent in the free wall of the right ventricle. 1 Recent studies have linked ARVC with mutations in proteins of the cardiac desmosome, 2 a component of the intercalated disc essential for mechanical coupling between cardiac cells. 3 It is estimated that as many as 70% of the mutations linked to familial ARVC are in the gene coding for plakophilin (PKP)2, 4 a 98-kDa desmosomal protein. PKP2 interacts with plakoglobin, desmoplakin, and the desmosomal cadherins via its amino terminal ("head") domain. [5][6] Loss of PKP2 destabilizes the desmosome, 7 and its genetic deletion in mice leads to rupture of the myocardial wall during the embryonic stage. 7 Loss of desmosomal integrity could lead to disruption of mechanical function in hearts afflicted with ARVC; yet, the latter does not directly explain the highly arrhythmogenic nature of the disease, particularly in cases in which lifethreatening arrhythmias occur in the absence of severe displacement of myocardium with fatty or fibrous tissue. 8 Recently, Saffitz and colleagues proposed that disruption of mechanical coupling may lead to loss of gap junctionmediated electrical communication between cells. 8 -10 This hypothesis awaits confirmation in a cellular model in which protein expression can be manipulated and intercellular communication can be assessed directly.Here, we used small interfering (si)RNA technology to silence PKP2 expression in neonatal cardiac cells, and we explored the effect of loss of PKP2 expression on the distribution and function of gap junctions. Our studies focused primarily on 2 cell populations: cardiac myocytes and epicardium-derived cells (EPDCs). Although the importance of cardiac myocytes in the context of ARVC and arrhythmias seems self-evident, a possible role for EPDCs in ARVC has not been described. Yet, as progenitors of the cardiac fibroblast cell lineage, the function of EPDCs deserves atte...

The cardiac electrical impulse depends on an orchestrated interplay of transmembrane ionic currents in myocardial cells. Two critical ionic current mechanisms are the inwardly rectifying potassium current (I K1 ), which is important for maintenance of the cell resting membrane potential, and the sodium current (I Na ), which provides a rapid depolarizing current during the upstroke of the action potential. By controlling the resting membrane potential, I K1 modifies sodium channel availability and therefore, cell excitability, action potential duration, and velocity of impulse propagation. Additionally, I K1 -I Na interactions are key determinants of electrical rotor frequency responsible for abnormal, often lethal, cardiac reentrant activity. Here, we have used a multidisciplinary approach based on molecular and biochemical techniques, acute gene transfer or silencing, and electrophysiology to show that I K1 -I Na interactions involve a reciprocal modulation of expression of their respective channel proteins (Kir2.1 and Na V 1.5) within a macromolecular complex. Thus, an increase in functional expression of one channel reciprocally modulates the other to enhance cardiac excitability. The modulation is model-independent; it is demonstrable in myocytes isolated from mouse and rat hearts and with transgenic and adenoviral-mediated overexpression/silencing. We also show that the post synaptic density, discs large, and zonula occludens-1 (PDZ) domain protein SAP97 is a component of this macromolecular complex. We show that the interplay between Na v 1.5 and Kir2.1 has electrophysiological consequences on the myocardium and that SAP97 may affect the integrity of this complex or the nature of Na v 1.5-Kir2.1 interactions. The reciprocal modulation between Na v 1.5 and Kir2.1 and the respective ionic currents should be important in the ability of the heart to undergo self-sustaining cardiac rhythm disturbances.reentry | scaffolding proteins | conduction velocity | protein trafficking I n the heart, the inward rectifying potassium current (I K1 ) is the major current responsible for the maintenance of the resting membrane potential (RMP), whereas the sodium current (I Na ) provides the largest fraction of the inward depolarizing current that flows during an action potential (1). It is well-known that a relationship exists between these two ionic currents that is crucial for proper cardiac electrical function; disruption of this balance results in changes in sodium channel availability, cell excitability, action potential duration, and conduction velocity (2). Accordingly, I K1 -I Na interactions are important in stabilizing and controlling the frequency of the electrical rotors that are responsible for the most dangerous cardiac arrhythmias, including ventricular tachycardia and fibrillation (3).Post synaptic density, discs large, and zonula occludens-1 (PDZ) domain proteins link different and in many cases, multiple proteins to macromolecular complexes through interactions with their various domains. More than 70 PDZ d...

Background Little is known about the mechanisms underlying the transition from paroxysmal to persistent atrial fibrillation (AF). In an ovine model of long-standing persistent AF (LS-PAF) we tested the hypothesis that the rate of electrical and/or structural remodeling, assessed by dominant frequency (DF) changes, determines the time at which AF becomes persistent. Methods and Results Self-sustained AF was induced by atrial tachypacing. Seven sheep were sacrificed 11.5±2.3 days after the transition to persistent AF and without reversal to sinus rhythm (SR); 7 sheep were sacrificed after 341.3±16.7 days of LS-PAF. Seven sham-operated animals were in SR for 1 year. DF was monitored continuously in each group. RT-PCR, western blotting, patch-clamping and histological analyses were used to determine changes in functional ion channel expression and structural remodeling. Atrial dilatation, mitral valve regurgitation, myocyte hypertrophy, and atrial fibrosis occurred progressively and became statistically significant after the transition to persistent AF, with no evidence for left ventricular dysfunction. DF increased progressively during the paroxysmal-to-persistent AF transition and stabilized when AF became persistent. Importantly, the rate of DF increase (dDF/dt) correlated strongly with the time to persistent AF. Significant action potential duration (APD) abbreviation, secondary to functional ion channel protein expression changes (CaV1.2, NaV1.5 and KV4.2 decrease; Kir2.3 increase), was already present at the transition and persisted for one-year follow up. Conclusions In the sheep model of LS-PAF, the rate of DF increase predicts the time at which AF stabilizes and becomes persistent, reflecting changes in APD and densities of sodium, L-type calcium and inward rectifier currents.

Objectives To determine whether Gal-3 mediates sustained atrial fibrillation (AF)-induced atrial structural and electrical remodeling and contributes to AF perpetuation. Background Galectin-3 (Gal-3) mediates extracellular matrix remodeling in heart failure, but its role in AF progression remains unexplored. Methods We examined intracardiac blood samples from patients with AF (N=55) to identify potential biomarkers of AF recurrence. In a sheep model of tachypacing-induced AF (N=20), we tested the effects of Gal-3 inhibition during AF progression. Results In patients, intracardiac serum Gal-3 levels were greater in persistent than paroxysmal AF and independently predicted atrial tachyarrhythmia recurrences after a single ablation procedure. In the sheep model, both Gal-3 and TGF-β1 were elevated in the atria of persistent AF animals. The Gal-3 inhibitor GM-CT-01 (GMCT) reduced both Gal-3 and TGF-β1-induced sheep atrial fibroblast migration and proliferation in vitro. GMCT (12 mg/kg twice/week) prevented the increase in serum procollagen type III N-terminal peptide seen during progression to persistent AF, and also mitigated atrial dilatation, myocyte hypertrophy, fibrosis, and the expected increase in dominant frequency of excitation. Atria of GMCT-treated animals had significantly less TGF-β1-Smad2/3 signaling pathway activation and expression of α-smooth muscle actin and collagen than saline-treated animals. Ex-vivo hearts from GMCT-treated animals had significantly longer action potential durations and fewer rotors and wavebreaks during AF, and myocytes had lower functional expression of inward rectifier K+ channel (Kir2.3) than saline-treated animals. Importantly, GMCT increased the probability of spontaneous AF termination, decreased AF inducibility and reduced overall AF burden. Conclusions Inhibiting Gal-3 during AF progression might be useful as an adjuvant treatment to improve outcomes of catheter ablation for persistent AF. Gal-3 inhibition may be a potential new upstream therapy for prevention of AF progression.

Background Voltage-gated Na+ channels (Nav) are essential for myocyte membrane excitability and cardiac function. Nav current (INa) is a large amplitude, short duration “spike” generated by rapid channel activation followed immediately by inactivation. However, even under normal conditions, a small “late” component of INa (INa,L) persists due to incomplete/failed inactivation of a subpopulation of channels. Notably, INa,L is directly linked with both congenital and acquired disease states. The multifunctional Ca2+/calmodulin-dependent kinase II (CaMKII) has been identified as an important activator of INa,L in disease. Several potential CaMKII phosphorylation sites have been discovered, including Ser571 in the Nav1.5 DI-DII linker, but the molecular mechanism underlying CaMKII-dependent regulation of INa,L in vivo remains unknown. Methods and Results To determine the in vivo role of Ser571, two Scn5a knock-in mouse models were generated expressing either: 1) Nav1.5 with a phosphomimetic mutation at Ser571 (S571E), or 2) Nav1.5 with the phosphorylation site ablated (S571A). Electrophysiology studies revealed that Ser571 regulates INa,L but not other channel properties previously linked to CaMKII. Ser571-mediated increases in INa,L promote abnormal repolarization and intracellular Ca2+ handling, and increase susceptibility to arrhythmia at the cellular and animal level. Importantly, Ser571 is required for maladaptive remodeling and arrhythmias in response to pressure overload. Conclusions Our data provide the first in vivo evidence for the molecular mechanism underlying CaMKII activation of the pathogenic INa,L. Relevant for improved rational design of potential therapies, our findings demonstrate that Ser571-dependent regulation of Nav1.5 specifically tunes INa,L without altering critical physiological components of the current.

Background Reduced Connexin43 (Cx43), sodium channel (Nav1.5) expression and increased collagen expression (fibrosis) are important determinants of impulse conduction in the heart. Objective To study the importance and interaction of these factors at very low Cx43 expression, inducible Cx43 KO mice with and without inducible ventricular tachycardia (VT) were compared by electrophysiology and immunohistochemistry. Methods Cx43CreER(T)/fl mice were induced with Tamoxifen and sacrificed after 2 weeks. Epicardial activation mapping was performed on Langendorff-perfused hearts, and arrhythmia vulnerability was tested. Mice were subdivided in VT+ (n=13) and VT− (n=10) and heart tissue was analyzed for Cx43, Nav1.5 and fibrosis. Results VT+ mice had decreased Cx43 expression with increased global, but not local, heterogeneity of Cx43, compared to VT− mice. Nav1.5-immunoreactive protein expression was reduced in VT+ versus VT− mice, specifically at sites devoid of Cx43. Levels of fibrosis were similar between VT− and VT+ mice. QRS-duration was increased and epicardial activation was more dispersed in VT+ mice than in VT− mice. The effective refractory period (ERP) was similar between both groups. Premature stimulation resulted in a more severe conduction slowing in VT+ compared to VT− hearts in the right ventricle. Separate patch clamp experiments in isolated rat ventricular myocytes confirmed that loss of Cx43 expression correlated with decreased sodium current amplitude. Conclusions Global heterogeneity in Cx43 expression and concomitant heterogeneous downregulation of sodium channel protein expression and sodium current leads to slowed and dispersed conduction, which sensitizes the heart for ventricular arrhythmias.