Faculty Bibliography

Protein histidine phosphatase 1 (PHPT-1) is an evolutionarily conserved 14 kDa protein that dephosphorylates phosphohistidine.PHPT-1

ATP-sensitive K+ (KATP) channels uniquely link cellular energy metabolism to membrane excitability and are expressed in diverse cell types that range from the endocrine pancreas to neurons and smooth, skeletal, and cardiac muscle. A decrease in the surface expression of KATP channels has been linked to various disorders, including dysregulated insulin secretion, abnormal blood pressure, and impaired resistance to cardiac injury. In contrast, up-regulation of KATP channel surface expression may be protective, for example, by mediating the beneficial effect of ischemic preconditioning. Molecular mechanisms that regulate KATP channel trafficking are poorly understood. Here, we used cellular assays with immunofluorescence, surface biotinylation, and patch clamping to demonstrate that Eps15 homology domain-containing protein 2 (EHD2) is a novel positive regulator of KATP channel trafficking to increase surface KATP channel density. EHD2 had no effect on cardiac Na+ channels (Nav1.5). The effect is specific to EHD2 as other members of the EHD family-EHD1, EHD3, and EHD4-had no effect on KATP channel surface expression. EHD2 did not directly affect KATP channel properties as unitary conductance and ATP sensitivity were unchanged. Instead, we observed that the mechanism by which EHD2 increases surface expression is by stabilizing KATP channel-containing caveolar structures, which results in a reduced rate of endocytosis. EHD2 also regulated KATP channel trafficking in isolated cardiomyocytes, which validated the physiologic relevance of these observations. Pathophysiologically, EHD2 may be cardioprotective as a dominant-negative EHD2 mutant sensitized cardiomyocytes to ischemic damage. Our findings highlight EHD2 as a potential pharmacologic target in the treatment of diseases with KATP channel trafficking defects.-Yang, H. Q., Jana, K., Rindler, M. J., Coetzee, W. A. The trafficking protein, EHD2, positively regulates cardiac sarcolemmal KATP channel surface expression: role in cardioprotection.

BACKGROUND:Genetic variant interpretation contributes to testing yield differences reported for sudden unexplained death. Adapting a high-resolution variant interpretation framework, which considers disease prevalence, reduced penetrance, genetic heterogeneity, and allelic contribution to determine the maximum tolerated allele count in gnomAD, we report an evaluation of cardiac channelopathy and cardiomyopathy genes in a large, demographically diverse sudden unexplained death cohort that underwent thorough investigation in the United States' largest medical examiner's office. METHODS AND RESULTS/RESULTS:The cohort has 296 decedents: 147 Blacks, 64 Hispanics, 49 Whites, 22 Asians, and 14 mixed ethnicities; 142 infants (1 to 11 months), 39 children (1 to 17 years), 74 young adults (18 to 34 years), and 41 adults (35 to 55 years). Eighty-nine cardiac disease genes were evaluated. Using a high-resolution variant interpretation workflow, we classified 17 variants as pathogenic or likely pathogenic (2 of which were incidental findings and excluded in testing yield analysis), 46 novel variants of uncertain significance, and 130 variants of uncertain significance. Nine pathogenic or likely pathogenic variants in ClinVar were reclassified to likely benign and excluded in testing yield analysis. The yields of positive cases by ethnicity and age were 21.4% in mixed ethnicities, 10.2% Whites, 4.5% Asians, 3.1% Hispanics, and 2% Blacks; 7.7% children, 7.3% in adults, 5.4% young adults, and 2.8% infants. The percentages of uncertain cases with variants of uncertain significance by ethnicity were 45.5% in Asians, 45.3% Hispanics, 44.20% Blacks, 36.7% Whites, and 14.3% in mixed ethnicities. CONCLUSIONS:High-resolution variant interpretation provides diagnostic accuracy and healthcare efficiency. Under-represented populations warrant greater inclusion in future studies.

Plakophilin-2 (PKP2) is a component of the desmosome and known for its role in cell-cell adhesion. Mutations in human PKP2 associate with a life-threatening arrhythmogenic cardiomyopathy, often of right ventricular predominance. Here, we use a range of state-of-the-art methods and a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mouse to demonstrate that in addition to its role in cell adhesion, PKP2 is necessary to maintain transcription of genes that control intracellular calcium cycling. Lack of PKP2 reduces expression of Ryr2 (coding for Ryanodine Receptor 2), Ank2 (coding for Ankyrin-B), Cacna1c (coding for CaV1.2) and Trdn (coding for triadin), and protein levels of calsequestrin-2 (Casq2). These factors combined lead to disruption of intracellular calcium homeostasis and isoproterenol-induced arrhythmias that are prevented by flecainide treatment. We propose a previously unrecognized arrhythmogenic mechanism related to PKP2 expression and suggest that mutations in PKP2 in humans may cause life-threatening arrhythmias even in the absence of structural disease.It is believed that mutations in desmosomal adhesion complex protein plakophilin 2 (PKP2) cause arrhythmia due to loss of cell-cell communication. Here the authors show that PKP2 controls the expression of proteins involved in calcium cycling in adult mouse hearts, and that lack of PKP2 can cause arrhythmia in a structurally normal heart.

BACKGROUND: Two genetic variants in SCN5A, encoding the Nav1.5 Na+ channel alpha-subunit, were found in a five month-old girl who died suddenly in her sleep. The first variant is a missense mutation, resulting in an amino acid change (Q1832E), which has been described (but not characterized) in a patient with Brugada syndrome. The second is a nonsense mutation that produces a premature stop codon and a C-terminal truncation (R1944Delta). METHODS AND RESULTS: To investigate their functional relevance with patch clamp experiments in transfected HEK293 cells. The Q1832E mutation drastically reduced Nav1.5 current density. The R1944Delta C-terminal truncation had negligible effects on Nav1.5 current density. Neither of the mutations affected the voltage dependence of steady activation and inactivation or influenced the late Na+ current or the recovery from inactivation. Biochemical and immunofluorescent approaches demonstrated that the Q1832E mutation caused severe trafficking defects. PCR cloning and sequencing the victim's genomic DNA allowed us to determine that the two variants were in trans. We investigated the functional consequences by co-expressing Nav1.5(Q1832E) and Nav1.5(R1944Delta), which led to a significantly reduced current amplitude relative to wild-type. CONCLUSIONS: These SIDS-related variants caused a severely dysfunctional Nav1.5 channel, which was mainly due to trafficking defects caused by the Q1832E mutation. The decreased current density is likely to be a major contributing factor to arrhythmogenesis in Brugada syndrome and the sudden death of this SIDS victim

AIMS: Myocardial ischemia remains the prime cause of morbidity and mortality in the United States. Ischemic preconditioning (IPC) is a powerful form of endogenous protection against myocardial infarction. We studied alterations in KATPchannels surface density as a potential mechanism of IPC's protection. METHODS AND RESULTS: Using cardiac-specific knockout of Kir6.2 subunits, we demonstrate an essential role for sarcolemmal KATPchannels in the infarct-limiting effect of IPC in the mouse heart. With biochemical membrane fractionation, we demonstrated that sarcolemmal KATPchannel subunits are distributed both to the sarcolemma and intracellular endosomal compartments. Global ischemia causes a loss of sarcolemmal KATPchannel subunit distribution and internalization to endosomal compartments. Ischemia-induced internalization of KATPchannels was prevented by CaMKII inhibition. KATPchannel subcellular redistribution was also observed with immunohistochemistry. Ischemic preconditioning prior to the index ischemia reduces not only the infarct size, but also prevents KATPchannel internalization. Furthermore, not only did adenosine mimic IPC by preventing infarct size, but it also prevented ischemia-induced KATPchannel internalization via a PKC-mediated pathway. We show that preventing endocytosis with dynasore reduces both KATPchannel internalization and strongly mitigates infarct development. CONCLUSIONS: Our data demonstrate that plasticity of KATPchannel surface expression must be considered as a potentially important mechanism of the protective effects of IPC and adenosine.

Sarcolemmal ATP-sensitive potassium (KATP) channels control skeletal muscle energy use through their ability to adjust membrane excitability and related cell functions in accordance with cellular metabolic status. Mice with disrupted skeletal muscle KATP channels exhibit reduced adipocyte size and increased fatty acid release into the circulation. As yet, the molecular mechanisms underlying this link between skeletal muscle KATP channel function and adipose mobilization have not been established. Here, we demonstrate that skeletal muscle-specific disruption of KATP channel function in transgenic (TG) mice promotes production and secretion of musclin. Musclin is a myokine with high homology to atrial natriuretic peptide (ANP) that enhances ANP signaling by competing for elimination. Augmented musclin production in TG mice is driven by a molecular cascade resulting in enhanced acetylation and nuclear exclusion of the transcription factor forkhead box O1 (FOXO1) - an inhibitor of transcription of the musclin encoding gene. Musclin production/secretion in TG is paired with increased mobilization of fatty acids and a clear trend toward increased circulating ANP, an activator of lipolysis. These data establish KATP channel-dependent musclin production as a potential mechanistic link coupling "local" skeletal muscle energy consumption with mobilization of bodily resources from fat. Understanding such mechanisms is an important step toward designing interventions to manage metabolic disorders including those related to excess body fat and associated co-morbidities.

KATP channels are integral to the functions of many cells and tissues. The use of electrophysiological methods has allowed for a detailed characterization of KATP channels in terms of their biophysical properties, nucleotide sensitivities, and modification by pharmacological compounds. However, even though they were first described almost 25 years ago (Noma 1983, Trube and Hescheler 1984), the physiological and pathophysiological roles of these channels, and their regulation by complex biological systems, are only now emerging for many tissues. Even in tissues where their roles have been best defined, there are still many unanswered questions. This review aims to summarize the properties, molecular composition, and pharmacology of KATP channels in various cardiovascular components (atria, specialized conduction system, ventricles, smooth muscle, endothelium, and mitochondria). We will summarize the lessons learned from available genetic mouse models and address the known roles of KATP channels in cardiovascular pathologies and how genetic variation in KATP channel genes contribute to human disease.

With advanced aging, there is a decline in innate cardiovascular function. This decline is not general in nature. Instead, specific changes occur that impact the basic cardiovascular function, which include alterations in biochemical pathways and ion channel function. This review focuses on a particular ion channel that couple the latter two processes, namely the KATP channel, which opening is promoted by alterations in intracellular energy metabolism. We show that the intrinsic properties of the KATP channel changes with advanced aging and argue that the channel can be further modulated by biochemical changes. The importance is widespread, given the ubiquitous nature of the KATP channel in the cardiovascular system where it can regulate processes as diverse as cardiac function, blood flow and protection mechanisms against superimposed stress, such as cardiac ischemia. We highlight questions that remain to be answered before the KATP channel can be considered as a viable target for therapeutic intervention.