The Cardiac Calcium Handling Machinery is Remodeled in Friedreich's Ataxia.

Background: Friedreich's ataxia (FA) is an inherited neurodegenerative disorder that causes progressive nervous system damage resulting in impaired muscle coordination. FA is the most common autosomal recessive form of ataxia and is caused by an expansion of the DNA triplet guanine-adenine-adenine (GAA) in the first intron of the Frataxin gene (FXN), located on chromosome 9q13. In the unaffected population, the number of GAA repeats ranges from 6 to 27 repetitions. In FA patients, GAA repeat expansions range from 44 to 1,700 repeats which decreases frataxin protein expression. Frataxin is a mitochondrial protein essential for various cellular functions, including iron metabolism. Reduced frataxin expression is thought to negatively affect mitochondrial iron metabolism, leading to increased oxidative damage. Although FA is considered a neurodegenerative disorder, FA patients display heart disease that includes hypertrophy, heart failure, arrhythmias, conduction abnormalities, and cardiac fibrosis. Objective: In this work, we investigated whether abnormal Ca 2+ handling machinery is the molecular mechanism that perpetuates cardiac dysfunction in FA. Methods: We used the frataxin knock-out (FXN-KO) mouse model of FA as well as human heart samples from donors with FA and from unaffected donors. ECG and echocardiography were used to assess cardiac function in the mice. Expression of calcium handling machinery proteins was assessed with proteomics and western blot. In left ventricular myocytes from FXN-KO and FXN-WT mice, the IonOptix system was used for calcium imaging, the seahorse assay was utilized to measure oxygen consumption rate (OCR), and confocal imaging was used to quantify the mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS). Results: We found that major contractile proteins, including SERCA2a and Ryr2, were downregulated in human left ventricular samples from deceased donors with FA compared to unaffected donors, similar to the downregulation of these proteins in the left ventricular tissue from FXN-KO compared to FXN-WT. On the ECG, the RR, PR, QRS, and QTc were significantly longer in the FXN-KO mice compared to FXN-WT. The ejection fraction and fractional shortening were significantly decreased and left ventricular wall thickness and diameter were significantly increased in the FXN-KO mice versus FXN-WT. The mitochondrial membrane potential Δψm was depolarized, ROS levels were elevated, and OCR was decreased in ventricular myocytes from FXN-KO versus FXN-WT. Conclusion: The development of left ventricular contractile dysfunction in FA is associated with reduced expression of calcium handling proteins and mitochondrial dysfunction.

Bioengineered peptibodies as blockers of ion channels.

We engineered and produced an ion channel blocking peptibody, that targets the acetylcholine-activated inwardly rectifying potassium current (IKACh). Peptibodies are chimeric proteins generated by fusing a biologically active peptide with the fragment crystallizable (Fc) region of the human immunoglobulin G (IgG). The IKACh blocking peptibody was engineered as a fusion between the human IgG1 Fc fragment and the IKACh inhibitor tertiapinQ (TP), a 21-amino acid synthetic peptidotoxin, originally isolated from the European honey bee venom. The peptibody was purified from the culture supernatant of human embryonic kidney (HEK) cells transfected with the peptibody construct. We tested the hypothesis that the bioengineered peptibody is bioactive and a potent blocker of IKACh. In HEK cells transfected with Kir3.1 and Kir3.4, the molecular correlates of IKACh, patch clamp showed that the peptibody was ~300-fold more potent than TP. Molecular dynamics simulations suggested that the increased potency could be due to an increased stabilization of the complex formed by peptibody-Kir3.1/3.4 channels compared to tertiapin-Kir3.1/3.4 channels. In isolated mouse myocytes, the peptibody blocked carbachol (Cch)-activated IKACh in atrial cells but did not affect the potassium inwardly rectifying background current in ventricular myocytes. In anesthetized mice, the peptibody abrogated the bradycardic effects of intraperitoneal Cch injection. Moreover, in aged mice, the peptibody reduced the inducibility of atrial fibrillation, likely via blocking constitutively active IKACh. Bioengineered anti-ion channel peptibodies can be powerful and highly potent ion channel blockers, with the potential to guide the development of modulators of ion channels or antiarrhythmic modalities.

The Arf6/PIP5K pathway activates IKACh in cigarette smoke mediated atrial fibrillation.

Cigarette smoking (CS) is a major cause of cardiovascular diseases. Smokers are at a significantly higher risk for developing atrial fibrillation (AF), a dangerous and abnormal heart rhythm. In the US, 15.5% of adults are current smokers, and it is becoming clear that CS is an independent risk factor for AF, but a detailed mechanistic understanding of how CS contributes to the molecular patho-electrophysiology of AF remains elusive. We investigated if CS related AF is in part mediated through a mechanism that depends on the cardiac acetylcholine activated inward rectifier potassium current (IKACh). We tested the hypothesis that CS increases IKACh via phosphatidylinositol 4-phosphate 5-kinase alpha (PIP5K) and ADP ribosylation factor 6 (Arf6) signaling, leading to AF perpetuation. In vivo inducibility of AF was assessed in mice exposed to CS for 8 weeks. AF duration was increased in CS exposed mice, and TertiapinQ, an IKACh blocker prevented AF development in CS exposed mice. In HEK293 cells stably transfected with Kir3.1 and Kir3.4, the molecular correlates of IKACh, CS exposure increased the expression of the Kir3.1 and Kir3.4 proteins at the cell surface, activated Arf6 and increased the IKACh current. Inhibition of PIP5K, or of Kir3.1/Kir3.4 trafficking via Arf6 abrogated the CS effects on IKACh. Cigarette smoke modifies the atrial electrophysiological substrate, leading to arrhythmogenesis, in part, through IKACh activation via an Arf6/PIP5K dependent pathway.

A benzopyran with antiarrhythmic activity is an inhibitor of Kir3.1-containing potassium channels.

Atrial fibrillation (AF) is the most commonly diagnosed cardiac arrhythmia and is associated with increased morbidity and mortality. Currently approved AF antiarrhythmic drugs have limited efficacy and/or carry the risk of ventricular proarrhythmia. The cardiac acetylcholine activated inwardly rectifying K+ current (IKACh), composed of Kir3.1/Kir3.4 heterotetrameric and Kir3.4 homotetrameric channel subunits, is one of the best validated atrial-specific ion channels. Previous research pointed to a series of benzopyran derivatives with potential for treatment of arrhythmias, but their mechanism of action was not defined. Here, we characterize one of these compounds termed Benzopyran-G1 (BP-G1) and report that it selectively inhibits the Kir3.1 (GIRK1 or G1) subunit of the KACh channel. Homology modeling, molecular docking, and molecular dynamics simulations predicted that BP-G1 inhibits the IKACh channel by blocking the central cavity pore. We identified the unique F137 residue of Kir3.1 as the critical determinant for the IKACh-selective response to BP-G1. The compound interacts with Kir3.1 residues E141 and D173 through hydrogen bonds that proved critical for its inhibitory activity. BP-G1 effectively blocked the IKACh channel response to carbachol in an in vivo rodent model and displayed good selectivity and pharmacokinetic properties. Thus, BP-G1 is a potent and selective small-molecule inhibitor targeting Kir3.1-containing channels and is a useful tool for investigating the role of Kir3.1 heteromeric channels in vivo. The mechanism reported here could provide the molecular basis for future discovery of novel, selective IKACh channel blockers to treat atrial fibrillation with minimal side effects.