Guadalupe Guerrero-Serna

Loss of Plakophilin-2 Expression Leads to Decreased Sodium Current and Slower Conduction Velocity in Cultured Cardiac Myocytes

Rationale: Plakophilin-2 (PKP2) is an essential component of the cardiac desmosome. Recent data show that it interacts with other molecules of the intercalated disc. Separate studies show preferential localization of the voltage-gated sodium channel (NaV1.5) to this region. Objective: To establish the association of PKP2 with sodium channels and its role on action potential propagation. Methods and Results: Biochemical, patch clamp, and optical mapping experiments demonstrate that PKP2 associates with NaV1.5, and that knockdown of PKP2 expression alters the properties of the sodium current, and the velocity of action potential propagation in cultured cardiomyocytes. Conclusions: These results emphasize the importance of intermolecular interactions between proteins relevant to mechanical junctions, and those involved in electric synchrony. Possible relevance to the pathogenesis of arrhythmogenic right ventricular cardiomyopathy is discussed.

Dominant Frequency Increase Rate Predicts Transition from Paroxysmal to Long-Term Persistent Atrial Fibrillation

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 we tested the hypothesis that the rate of electric and 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 euthanized 11.5±2.3 days after the transition to persistent AF and without reversal to sinus rhythm; 7 sheep were euthanized after 341.3±16.7 days of long-standing persistent AF. Seven sham-operated animals were in sinus rhythm for 1 year. DF was monitored continuously in each group. Real-time polymerase chain reaction, Western blotting, patch clamping, and histological analyses were used to determine the 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 correlated strongly with the time to persistent AF. Significant action potential duration 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 1 year of follow up. Conclusions— In the sheep model of long-standing persistent AF, the rate of DF increase predicts the time at which AF stabilizes and becomes persistent, reflecting changes in action potential duration and densities of sodium, L-type calcium, and inward rectifier currents.

Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes

Applications of human induced pluripotent stem cell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However, purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here, we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins, gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells, MLC-2v selected CMs had larger action potential amplitudes and durations. In addition, by immunofluorescence, we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricular myocyte lineages. However, only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach, it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers.

Designing Heart Performance by Gene Transfer

The birth of molecular cardiology can be traced to the development and implementation of high-fidelity genetic approaches for manipulating the heart. Recombinant viral vector-based technology offers a highly effective approach to genetically engineer cardiac muscle in vitro and in vivo. This review highlights discoveries made in cardiac muscle physiology through the use of targeted viral-mediated genetic modification. Here the history of cardiac gene transfer technology and the strengths and limitations of viral and nonviral vectors for gene delivery are reviewed. A comprehensive account is given of the application of gene transfer technology for studying key cardiac muscle targets including Ca(2+) handling, the sarcomere, the cytoskeleton, and signaling molecules and their posttranslational modifications. The primary objective of this review is to provide a thorough analysis of gene transfer studies for understanding cardiac physiology in health and disease. By comparing results obtained from gene transfer with those obtained from transgenesis and biophysical and biochemical methodologies, this review provides a global view of cardiac structure-function with an eye towards future areas of research. The data presented here serve as a basis for discovery of new therapeutic targets for remediation of acquired and inherited cardiac diseases.

TGF-β1, Released by Myofibroblasts, Differentially Regulates Transcription and Function of Sodium and Potassium Channels in Adult Rat Ventricular Myocytes

Cardiac injury promotes fibroblasts activation and differentiation into myofibroblasts, which are hypersecretory of multiple cytokines. It is unknown whether any of such cytokines are involved in the electrophysiological remodeling of adult cardiomyocytes. We cultured adult cardiomyocytes for 3 days in cardiac fibroblast conditioned medium (FCM) from adult rats. In whole-cell voltage-clamp experiments, FCM-treated myocytes had 41% more peak inward sodium current (INa) density at −40 mV than myocytes in control medium (p<0.01). In contrast, peak transient outward current (Ito) was decreased by ∼55% at 60 mV (p<0.001). Protein analysis of FCM demonstrated that the concentration of TGF-β1 was >3 fold greater in FCM than control, which suggested that FCM effects could be mediated by TGF-β1. This was confirmed by pre-treatment with TGF-β1 neutralizing antibody, which abolished the FCM-induced changes in both INa and Ito. In current-clamp experiments TGF-β1 (10 ng/ml) prolonged the action potential duration at 30, 50, and 90 repolarization (p<0.05); at 50 ng/ml it gave rise to early afterdepolarizations. In voltage-clamp experiments, TGF-β1 increased INa density in a dose-dependent manner without affecting voltage dependence of activation or inactivation. INa density was −36.25±2.8 pA/pF in control, −59.17±6.2 pA/pF at 0.1 ng/ml (p<0.01), and −58.22±6.6 pA/pF at 1 ng/ml (p<0.01). In sharp contrast, Ito density decreased from 22.2±1.2 pA/pF to 12.7±0.98 pA/pF (p<0.001) at 10 ng/ml. At 1 ng/ml TGF-β1 significantly increased SCN5A (NaV1.5) (+73%; p<0.01), while reducing KCNIP2 (Kchip2; −77%; p<0.01) and KCND2 (KV4.2; −50% p<0.05) mRNA levels. Further, the TGF-β1-induced increase in INa was mediated through activation of the PI3K-AKT pathway via phosphorylation of FOXO1 (a negative regulator of SCN5A). TGF-β1 released by myofibroblasts differentially regulates transcription and function of the main cardiac sodium channel and of the channel responsible for the transient outward current. The results provide new mechanistic insight into the electrical remodeling associated with myocardial injury.

Extracellular Matrix–Mediated Maturation of Human Pluripotent Stem Cell–Derived Cardiac Monolayer Structure and Electrophysiological Function

Background—Human pluripotent stem cell–derived cardiomyocytes (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 electric impulse propagation velocity and immature action potential profiles. Methods and Results—Here, we have identified an optimal extracellular matrix for significant electrophysiological and structural maturation of hPSC-CM monolayers. hPSC-CM plated in the optimal extracellular matrix combination have impulse propagation velocities ≈2× faster than previously reported (43.6±7.0 cm/s; 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 extracellular matrix promoted hypertrophic growth of cardiomyocytes and the expression of key mature sarcolemmal (SCN5A, Kir2.1, and connexin43) and myofilament markers (cardiac troponin I). The maturation process reported here relies on activation of integrin signaling pathways: neutralization of &bgr;1 integrin receptors via blocking antibodies and pharmacological blockade of focal adhesion kinase activation prevented structural maturation. Conclusions—Maturation of human stem cell–derived cardiomyocyte monolayers is achieved in a 1-week period by plating cardiomyocytes on PDMS (polydimethylsiloxane) coverslips rather than on conventional 2-dimensional cell culture formats, such as glass coverslips or plastic dishes. Activation of integrin signaling and focal adhesion kinase is essential for significant maturation of human cardiac monolayers.

Ca2+-independent positive molecular inotropy for failing rabbit and human cardiac muscle by α-myosin motor gene transfer

Current inotropic therapies used to increase cardiac contractility of the failing heart center on increasing the amount of calcium available for contraction, but their long‐term use is associated with increased mortality due to fatal arrhythmias. Thus, there is a need to develop and explore novel inotropic therapies that can act via calcium‐independent mechanisms. The purpose of this study was to determine whether fast α‐myosin molecular motor gene transfer can confer calcium‐independent positive inotropy in slow β‐myosin‐dominant rabbit and human failing ventricular myocytes. To this end, we generated a recombinant adenovirus (AdMYH6) to deliver the full‐length human α‐myosin gene to adult rabbit and human cardiac myocytes in vitro. Fast α‐myosin motor expression was determined by Western blotting and immunocytochemical analysis and confocal imaging. In experiments using electrically stimulated myocytes from ischemic failing hearts, AdMYH6 increased the contractile amplitude of failing human [23.9±7.8 nm (n=10) vs. AdMYH6 amplitude 78.4±16.5 nm (n=6)] and rabbit myocytes. The intracellular calcium transient amplitude was not altered. Control experiments included the use of a green fluorescent protein or a β‐myosin heavy chain adenovirus. Our data provide evidence for a novel form of calcium‐independent positive inotropy in failing cardiac myocytes by fast α‐myosin motor protein gene transfer.—Herron, T. J., Devaney, E., Mundada, L., Arden, E., Day, S., Guerrero‐Serna, G., Turner, I., Westfall, M., Metzger, J. M. Ca2+‐independent positive molecular inotropy for failing rabbit and human cardiac muscle by α‐myosin motor gene transfer. FASEB J. 24, 415–424 (2010). www.fasebj.org

Inhibition of platelet-derived growth factor-AB signaling prevents electromechanical remodeling of adult atrial myocytes that contact myofibroblasts

BACKGROUND Persistent atrial fibrillation (PAF) results in electromechanical and structural remodeling by mechanisms that are poorly understood. Myofibroblast proliferation and fibrosis are major sources of structural remodeling in PAF. Myofibroblasts also interact with atrial myocytes via direct physical contact and release of signaling molecules, which may contribute to remodeling. OBJECTIVE To determine whether myofibroblasts contribute to atrial myocyte electromechanical remodeling via direct physical contact and platelet-derived growth factor (PDGF) signaling. METHODS Myofibroblasts and myocytes from adult sheep atria were co-cultured for 24 hours. Alternatively adult sheep atrial myocytes were exposed to 1 ng/mL recombitant PDGF AB peptide for 24 hours. RESULTS Myocytes making contact with myofibroblasts demonstrated significant reduction (P ≤ .05) in peak L-type calcium current density, shortening of action potential duration (APD), and reduction in calcium transients. These effects were blocked by pretreatment with a PDGF-AB neutralizing anti-body. Heterocellular contact also severely disturbed the localization of the L-type calcium channel. Myocytes exposed to recombinant PDGF-AB peptide for 24 hours demonstrated reduced APD50, APD80 and Peak L-type calcium current. Pretreatment with a PDGF-AB neutralizing antibody prevented these effects. Finally, while control atrial myocytes did not respond in a 1:1 manner to pacing frequencies of 3 Hz or higher, atrial myocytes from hearts that were tachypaced for 2 months and normal myocytes treated with PDGF-AB for 24 hours could be paced up to 10 Hz. CONCLUSIONS In addition to leading to fibrosis, atrial myofibroblasts contribute to electromechanical remodeling of myocytes via direct physical contact and release of PDGF-AB, which may be a factor in PAF-induced remodeling.

Galectin-3 Regulates Atrial Fibrillation Remodeling and Predicts Catheter Ablation Outcomes

Summary Atrial fibrillation (AF) usually starts as paroxysmal but can evolve relentlessly to the persistent and permanent forms. However, the mechanisms governing such a transition are unknown. The authors show that intracardiac serum levels of galectin (Gal)-3 are greater in patients with persistent than paroxysmal AF and that Gal-3 independently predicts atrial tachyarrhythmia recurrences after a single ablation procedure. Using a sheep model of persistent AF the authors further demonstrate that upstream therapy targeting Gal-3 diminishes both electrical remodeling and fibrosis by impairing transforming growth factor beta–mediated signaling and reducing myofibroblast activation. Accordingly, Gal-3 inhibition therapy increases the probability of AF termination and reduces the overall burden of AF. Therefore the authors postulate that Gal-3 inhibition is a potential new upstream therapy to prevent AF progression.

Constitutive Intracellular Na+ Excess in Purkinje Cells Promotes Arrhythmogenesis at Lower Levels of Stress Than Ventricular Myocytes From Mice With Catecholaminergic Polymorphic Ventricular Tachycardia.

Background— In catecholaminergic polymorphic ventricular tachycardia (CPVT), cardiac Purkinje cells (PCs) appear more susceptible to Ca2+ dysfunction than ventricular myocytes (VMs). The underlying mechanisms remain unknown. Using a CPVT mouse (RyR2R4496C+/Cx40eGFP), we tested whether PC intracellular Ca2+ ([Ca2+]i) dysregulation results from a constitutive [Na+]i surplus relative to VMs. Methods and Results— Simultaneous optical mapping of voltage and [Ca2+]i in CPVT hearts showed that spontaneous Ca2+ release preceded pacing-induced triggered activity at subendocardial PCs. On simultaneous current-clamp and Ca2+ imaging, early and delayed afterdepolarizations trailed spontaneous Ca2+ release and were more frequent in CPVT PCs than CPVT VMs. As a result of increased activity of mutant ryanodine receptor type 2 channels, sarcoplasmic reticulum Ca2+ load, measured by caffeine-induced Ca2+ transients, was lower in CPVT VMs and PCs than respective controls, and sarcoplasmic reticulum fractional release was greater in both CPVT PCs and VMs than respective controls. [Na+]i was higher in both control and CPVT PCs than VMs, whereas the density of the Na+/Ca2+ exchanger current was not different between PCs and VMs. Computer simulations using a PC model predicted that the elevated [Na+]i of PCs promoted delayed afterdepolarizations, which were always preceded by spontaneous Ca2+ release events from hyperactive ryanodine receptor type 2 channels. Increasing [Na+]i monotonically increased delayed afterdepolarization frequency. Confocal imaging experiments showed that postpacing Ca2+ spark frequency was highest in intact CPVT PCs, but such differences were reversed on saponin-induced membrane permeabilization, indicating that differences in [Na+]i played a central role. Conclusions— In CPVT mice, the constitutive [Na+]i excess of PCs promotes triggered activity and arrhythmogenesis at lower levels of stress than VMs.