Faculty Bibliography

In the last decade, there have been considerable advances in the understanding of the pathophysiology of malignant ventricular tachyarrhythmias (VT) and sudden cardiac death (SCD). Over 80% of SCD occurs in patients with organic heart disease. However, approximately 10%-15% of SCD occurs in the presence of structurally normal heart, and the majority of these patients are young. In this group of patients, changes in genes encoding cardiac ion channels produce modifications of the function of the channel resulting in an electrophysiological substrate of VT and SCD. Collectively, these disorders are referred to as cardiac ion channelopathies. The four major syndromes in this group are: the long QT syndrome (LQTS), the Brugada syndrome (BrS), the short QT syndrome (SQTS), and the catecholaminergic polymorphic ventricular tachycardia (CPVT). Each of these syndromes includes multiple subtypes with different and sometimes complex cardiac ion channel genetic abnormalities. Many are associated with other somatic and neurological abnormalities besides the risk of VT and SCD. The current management of cardiac ion channelopathies can be summarized as follows: (1) in symptomatic patients, the implantable cardioverter defibrillator (ICD) is the only viable option; (2) in asymptomatic patients, risk stratification is necessary, followed by either the ICD, pharmacotherapy, or a combination of both. A genotype-specific approach to pharmacotherapy requires a thorough understanding of the molecular-cellular basis of arrhythmogenesis in cardiac ion channelopathies as well as the specific drug profile.

BACKGROUND: -Emerging clinical evidence demonstrates high prevalence of QTc prolongation and complex ventricular arrhythmias in patients with anti-Ro antibody (anti-Ro Ab) positive autoimmune diseases. We tested the hypothesis that anti-Ro Abs target the HERG K+ channel which conducts the rapidly activating delayed K+ current, IKr, thereby causing delayed repolarization seen as QT interval prolongation on the electrocardiogram (ECG). METHODS AND RESULTS: -Anti-Ro Ab positive sera, purified IgG and affinity purified anti-52kDa Ro Abs from patients with autoimmune diseases and QTc prolongation were tested on IKr using HEK293 cells expressing HERG channel and native cardiac myocytes. Electrophysiological and biochemical data demonstrate that anti-Ro Abs inhibit IKr to prolong action potential duration by directly binding to the HERG channel protein. 52kDa Ro antigen immunized guinea-pigs showed QTc prolongation on ECG after developing high titers of anti-Ro Abs which inhibited native IKr and cross-reacted with guinea-pig ERG channel. CONCLUSIONS: -The data establish that anti-Ro Abs from patients with autoimmune diseases inhibit IKr by cross-reacting with the HERG channel likely at the pore region where homology between 52Ro antigen and HERG channel is present. The animal model of autoimmune-associated QTc prolongation is the first to provide strong evidence for a pathogenic role of anti-Ro Abs in the development of QTc prolongation. Together, it is proposed that adult patients with anti-Ro Abs may benefit from routine ECG screening and those with QTc prolongation should receive counselling about drugs that may increase the risk for life threatening arrhythmias.

Sphingomyelin synthase related protein (SMSr) synthesizes the sphingomyelin analog ceramide phosphoethanolamine (CPE) in cells. Previous cell studies indicated that SMSr is involved in ceramide homeostasis and is crucial for cell function. To furhter examine SMSr function in vivo, we generated Smsr KO mice which were fertile and had no obvious phenotypic alterations. Quantitative MS analyses of plasma, liver, and macrophages from the KO mice revealed only marginal changes in CPE and ceramide, as well as other sphingolipid levels. Because SMS2 also has CPE synthase activity, we prepared Smsr/Sms2 double KO mice. We found that CPE levels were not significantly changed in macrophages, suggesting that CPE levels are not exclusively dependent on SMSr and SMS2 activities. We then measured CPE levels in Sms1 KO mice and found that Sms1 deficiency also reduced plasma CPE levels. Importantly, we found that expression of Sms1 or Sms2 in SF9 insect cells not only significantly increased SM but also CPE formation, indicating that SMS1 also has CPE synthase activity. Our study reveals that all mouse SMS family members (SMSr, SMS1, and SMS2) have CPE synthase activity. However, neither CPE nor SMSr appears to be a critical regulator of ceramide levels in vivo.

Voltage gated sodium channels (Nav) are transmembrane proteins responsible for action potential initiation. Mutations mainly located in the voltage sensor domain (VSD) of Nav1.5, the cardiac sodium channel, have been associated with the development of arrhythmias combined with dilated cardiomyopathy. Gating pore currents have been observed with three unrelated mutations associated with similar clinical phenotypes. However, gating pores have never been associated with mutations outside the first domain of Nav1.5. The aim of this study was to explore the possibility that gating pore currents might be caused by the Nav1.5 R225P and R814W mutations (R3, S4 in DI and DII, respectively), which are associated with rhythm disturbances and dilated cardiomyopathy. Nav1.5 WT and mutant channels were transiently expressed in tsA201 cells. The biophysical properties of the alpha pore currents and the presence of gating pore currents were investigated using the patch-clamp technique. We confirmed the previously reported gain of function of the alpha pores of the mutant channels, which mainly consisted of increased window currents mostly caused by shifts in the voltage dependence of activation. We also observed gating pore currents associated with the R225P and R814W mutations. This novel permeation pathway was open under depolarized conditions and remained temporarily open at hyperpolarized potentials after depolarization periods. Gating pore currents could represent a molecular basis for the development of uncommon electrical abnormalities and changes in cardiac morphology. We propose that this biophysical defect be routinely evaluated in the case of Nav1.5 mutations on the VSD.

Intestinal cholesterol absorption involves chylomicron and HDL pathways and is dependent on MTP and ABCA1, respectively. Chylomicrons transport free and esterified cholesterol whereas HDL transport free cholesterol. ACAT2 esterifies cholesterol for secretion with chylomicrons. We hypothesized that free cholesterol accumulated during ACAT2 deficiency may be secreted with HDL when chylomicron assembly is blocked. To test this, we studied cholesterol absorption in mice deficient in intestinal MTP, global ACAT2, and both intestinal MTP and global ACAT2. Intestinal MTP ablation significantly increased intestinal triglyceride and cholesterol levels and reduced their transport with chylomicrons. In contrast, global ACAT2 deficiency had no effect on triglyceride absorption but significantly reduced cholesterol absorption with chylomicrons and increased cellular free cholesterol. Their combined deficiency reduced cholesterol secretion with both chylomicrons and HDL. Thus, contrary to our hypothesis, free cholesterol accumulated in the absence of MTP and ACAT2 is unavailable for secretion with HDL. Global ACAT2 deficiency causes mild hypertriglyceridemia and reduces hepatosteatosis in mice fed high cholesterol diets by increasing hepatic lipoprotein production by unknown mechanisms. We show that this phenotype is preserved in the absence of intestinal MTP in global ACAT2 deficient mice fed a Western diet. Further, we observed increases in hepatic MTP activity in these mice. Thus, ACAT2 deficiency might increase MTP expression to avoid hepatosteatosis in cholesterol-fed animals. Therefore, ACAT2 inhibition might avert hepatosteatosis associated with high cholesterol diets by increasing hepatic MTP expression and lipoprotein production.

The Summer Institute Program to Increase Diversity (SIPID) in Health-Related Research is a career advancement opportunity sponsored by the National Heart, Lung, and Blood Institute. Three mentored programs address difficulties experienced by junior investigators in establishing independent research careers and academic advancement. Aims are to increase the number of faculty from under-represented minority groups who successfully compete for external research funding. Data were collected using a centralized data-entry system from three Summer Institutes. Outcomes include mentees' satisfaction rating about the program, grant and publications productivity and specific comments. Fifty-eight junior faculty mentees (38% male) noticeably improved their rates of preparing/submitting grant applications and publications, with a 18-23% increase in confidence levels in planning and conducting research. According to survey comments, the training received in grantsmanship skills and one-on-one mentoring were the most valuable program components. The SIPID mentoring program was highly valued by the junior faculty mentees. The program will continue in 2011-2014 as PRIDE (PRogram to Increase Diversity among individuals Engaged in health-related research). Long-term follow-up of current mentees will be indexed at five years post training (2013). In summary, these mentoring programs hope to continue increasing the diversity of the next generation of scientists in biomedical research.

BACKGROUND: The neuroendocrine Cav1.3 L-type Ca channels have been recently found in the Human fetal heart and shown to play a vital role in Ca entry from the sarcolemma into the cell and in Ca homeostasis. Calreticulin, a Ca binding endoplasmic reticulum (ER) resident protein, has been recently shown to translocate to the cell surface where its role and function are just emerging. Here, we demonstrated a novel mechanism of Cav1.3 and calreticulin interaction resulting in downregulation of Cav1.3 channel densities in native Human fetal cardiac cells and Human Embryonic Kidney cell lines (tsA201). METHODS AND RESULTS: Cell surface and cytoplasmic staining of calreticulin was demonstrated first in cultured human fetal cardiomyocytes (HFC), gestational age 18-24weeks, using confocal microscopy thereby establishing that calreticulin is present at the cell surface in HFC. Co-immunoprecipitation from HFC using anti-Cav1.3 Ca channel antibody, and probing with anti-calreticulin antibody revealed a 46kDa band corresponding to calreticulin suggesting that Cav1.3 Ca channel and calreticulin co-assemble in a macromolecular complex. Co-expression of Cav1.3 and calreticulin in tsA201 cells resulted in a decrease in surface expression of Cav1.3 Ca channels. These findings were consistent with the electrophysiological studies showing that co-transfection of Cav1.3 Ca channel and calreticulin resulted in 55% reduction of Cav1.3 Ca current densities recorded from tsA201 cells. CONCLUSIONS: The results show the first evidence that calreticulin: (1) is localized outside the ER on the cell surface of HFC; (2) coimmunoprecipitates with Cav1.3 L-type Ca channel; (3) negatively regulates Cav1.3 surface expression thus resulting in decreased Cav1.3 Ca current densities. The data demonstrate a novel mechanism of modulation of Cav1.3 Ca channel by calreticulin, which may be involved in pathological settings such as autoimmune associated congenital heart block where Cav1.3 Ca channels are downregulated.