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 β1 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.

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 (Ca _V 1.2, Na _V 1.5, and K _V 4.2 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.

Rationale:: In cardiomyocytes, Na _V 1.5 and Kir2.1 channels interact dynamically as part of membrane bound macromolecular complexes. Objective:: The objective of this study was to test whether Na _V 1.5 and Kir2.1 preassemble during early forward trafficking and travel together to common membrane microdomains. Methods and Results:: In patch-clamp experiments, coexpression of trafficking-deficient mutants Kir2.1 ^Δ314-315 or Kir2.1 ^R44A/R46A with wild-type (WT) Na _V 1.5 ^WT in heterologous cells reduced inward sodium current compared with Na _V 1.5 ^WT alone or coexpressed with Kir2.1 ^WT . In cell surface biotinylation experiments, expression of Kir2.1 ^Δ314-315 reduced Na _V 1.5 channel surface expression. Glycosylation analysis suggested that Na _V 1.5 ^WT and Kir2.1 ^WT channels associate early in their biosynthetic pathway, and fluorescence recovery after photobleaching experiments demonstrated that coexpression with Kir2.1 increased cytoplasmic mobility of Na _V 1.5 ^WT , and vice versa, whereas coexpression with Kir2.1 ^Δ314-315 reduced mobility of both channels. Viral gene transfer of Kir2.1 ^Δ314-315 in adult rat ventricular myocytes and human induced pluripotent stem cell–derived cardiomyocytes reduced inward rectifier potassium current and inward sodium current, maximum diastolic potential and action potential depolarization rate, and increased action potential duration. On immunostaining, the AP1 (adaptor protein complex 1) colocalized with Na _V 1.5 ^WT and Kir2.1 ^WT within areas corresponding to t-tubules and intercalated discs. Like Kir2.1 ^WT , Na _V 1.5 ^WT coimmunoprecipitated with AP1. Site-directed mutagenesis revealed that Na _V 1.5 ^WT channels interact with AP1 through the Na _V 1.5 ^Y1810 residue, suggesting that, like for Kir2.1 ^WT , AP1 can mark Na _V 1.5 channels for incorporation into clathrin-coated vesicles at the trans -Golgi. Silencing the AP1 ϒ-adaptin subunit in human induced pluripotent stem cell–derived cardiomyocytes reduced inward rectifier potassium current, inward sodium current, and maximum diastolic potential and impaired rate-dependent action potential duration adaptation. Conclusions:: The Na _V 1.5-Kir2.1 macromolecular complex pre-assembles early in the forward trafficking pathway. Therefore, disruption of Kir2.1 trafficking in cardiomyocytes affects trafficking of Na _V 1.5, which may have important implications in the mechanisms of arrhythmias in inheritable cardiac diseases.

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 (I_Na) density at −40 mV than myocytes in control medium (p<0.01). In contrast, peak transient outward current (I_to) was decreased by ∼55% at 60 mV (p3 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 I_Na and I_to. 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 I_Na density in a dose-dependent manner without affecting voltage dependence of activation or inactivation. I_Na 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, I_to 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 (Na_V1.5) (+73%; p<0.01), while reducing KCNIP2 (Kchip2; −77%; p<0.01) and KCND2 (K_V4.2; −50% p<0.05) mRNA levels. Further, the TGF-β1-induced increase in I_Na 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.

Obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased prevalence due to the rise of sedentary lifestyles and excessive caloric intake from processed food rich in fat and sugar. There are several definitions of MetS, but most of them describe it as a cluster of cardiovascular and metabolic alterations such as abdominal obesity, reduced high-density lipoprotein (HDL) and elevated low-density lipoprotein (LDL) cholesterol, elevated triglycerides, glucose intolerance, and hypertension. Diagnosis requires three out of these five criteria to be present. Despite the increasing prevalence of MetS, the understanding of its pathophysiology and relationship with disease is still limited. Indeed, the pathological consequences of MetS components have been reported individually, but investigations that have studied the effect of the combination of MeS components on organ pathological remodeling are almost nonexistent. On the other hand, animal models are a powerful tool in understanding the mechanisms that underlie pathological processes such as MetS. In the first part of the review, we will briefly overview the advantages, disadvantages and pathological manifestations of MetS in porcine, canine, rodent, and rabbit diet-induced experimental models. Then, we will focus on the different dietary regimes that have been used in rabbits to induce MetS by means of high-fat, cholesterol, sucrose or fructose-enriched diets and their effects on physiological systems and organ remodeling. Finally, we will discuss the use of dietary regimes in different transgenic strains and special rabbit breeds.