No abstract

No abstract

Abstract-Amino acid sequence variations in SCN5A are known to affect function of wild-type channels and also those with coexisting mutations; therefore, it is important to know the exact sequence and function of channels most commonly present in human myocardium. SCN5A was analyzed in control panels of human alleles, demonstrating that the existing clones (hH1, hH1a, hH1b) each contained a rare variant and thus none represented the common sequence. Confirming prior work, the H558R polymorphism was present in Ϸ30% of subjects. Quantitative mRNA analysis from human hearts showed that a shorter 2015 amino acid splice variant lacking glutamine at position 1077 (Q1077del) made up 65% of the transcript in every heart examined. Age, sex, race, or structural heart disease did not affect this proportion of Q1077del. Estimated population frequencies for the four common variants were 25% SCN5A, 10% [H558R], 45% [Q1077del], and 20% [H558R;Q1077del], where the reference sequence SCN5A is GenBank AC137587. When expressed in HEK-293 cells, these common variants had a more positive mid-point of the voltage dependence of inactivation than the standard clone hH1. Also, channels containing Q1077 expressed smaller currents. When H558R was present with Q1077 ([H558R]), current expression was profoundly reduced despite normal trafficking to the cell surface. Thus, four variant sequences for SCN5A are commonly present in human myocardium and they exhibit functional differences among themselves and with the previous standard clone. SCN5A encodes the voltage-dependent sodium channel ␣-subunit protein SCN5A, also called hNa v 1.5, 1 found predominantly in human heart muscle. This channel is responsible for large peak inward sodium current (I Na ) that underlies excitability and conduction in working myocardium (atrial and ventricular cells) and special conduction tissue (Purkinje cells and others), and also for late I Na that influences repolarization and refractoriness. Three complete cDNA clones for this channel hH1, 2 hH1a, 3 and hH1b 4 differ in amino acid sequence in 5 of the 2016 positions (Table 1). In addition, these three clones differ from the deduced amino acid sequence for SCN5A obtained from the two human genome databases: Celera and the International Human Genome Sequencing Collaboration (IHGSC). Before the present study, it was not clear whether or not these differences are present in human population as common variants. From previous studies, we know that dramatic differences in current expression can be found when arrhythmia mutations are expressed in different background clones. 4 This study was designed to answer the questions: What is the common background sequence for SCN5A? Do common variations affect channel function? Does it matter which Na ϩ channel clone (ie, background sequence) is used for functional studies of wild-type and mutated channels? Materials and MethodsProtocols used in this investigation are more fully described in the expanded Materials and Methods section in the online data supplement available...

IntroductionK ATP channels respond to changes in the intracellular ATP content by altering a cell's membrane potential (1, 2). K ATP channels are widely expressed in neural, endocrine, and muscle tissues where they are inhibited by ATP and stimulated by ADP. K ATP channels are an octameric complex consisting of four potassium channel subunits, either Kir6.1 or Kir6.2, and four sulfonylurea receptor subunits, SUR1 or SUR2 (3-9). SURs, named for their ability to bind with high affinity the hypoglycemic sulfonylurea agents, are members of the ATP-binding cassette (ABC) transporter family of transmembrane proteins. SUR1 and SUR2 are 70% identical proteins encoded by different genes. SUR2 undergoes differential splicing altering its carboxy-terminal 42 amino acids, yielding channels with unique pharmacologic properties.In cardiomyocytes, where K ATP channels modulate protection from ischemia, SUR1 and SUR2 are coexpressed (10, 11). In smooth muscle and voluntary striated muscle, only SUR2 is expressed (11). The physiology and pharmacology of K ATP channels have been most extensively studied in the pancreatic β cell. Here, sulfonylurea agents such as glibenclamide inhibit K ATP channels by binding to SUR1, which results in the closure of the channel and the stimulation of insulin release (3). In vascular smooth muscle, potassium channel openers, such as nicorandil, implicate K ATP channels in the regulation of tonic vasomotor activity. These agents, useful in the treatment of hypertension and angina, open K ATP channels leading to potassium efflux, membrane hyperpolarization, and vasodilation (12-14). Potassium channel openers alter membrane potential through K ATP channels and thereby activate voltage-dependent calcium channels producing changes in vascular smooth muscle contractility (15). In skeletal muscle, K ATP channels affect glucose metabolism. Using mice with a targeted disruption of the Sur2 gene (16), we demonstrated that the loss of K ATP channels increased insulin responsiveness mediated by striated muscle.The diversity of responses to individual pharmacologic agents that act through K ATP channels derives in part from the tissue-specific expression of K ATP subunits and the composition of K ATP channels within a cell. Since pharmacologic agents act through the SUR subunit, the significant homology between SUR isoforms and their overlapping expression pattern complicates the interpretation of pharmacologic studies in This article was published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

Objective Recent studies have revealed that microRNAs (miRNAs) are involved in the regulation of cardiac development, physiologic, and pathologic processes via post-transcriptional control of gene expression. The stable circulating miRNAs offer unique opportunities for the early diagnosis of several diseases. In this study, we examined the circulating miR-133 and miR-328 levels from patients with acute myocardial infarction (AMI). Patients and Methods Twenty-eight control subjects and fifty-one consecutive AMI patients were enrolled. The plasma and whole blood samples from AMI patients were obtained within 24 hours (n=51) and 7 days (n=6) after the onset of AMI symptoms. The circulating miR-133 and miR-328 levels were analyzed using quantitative real-time PCR. Results The miR-133 levels in plasma from AMI patients exhibited a 4.4-fold increase compared with control subjects (p=0.006). Moreover, the increased miR-133 levels in whole blood were comparable with those in plasma samples. In contrast, the miR-328 levels in plasma and whole blood of AMI patients were markedly increased by 10.9-fold and 16.1-fold, respectively, compared to those in control subjects (p=0.033 and p <0.001). The elevated circulating miR-133 and miR-328 levels were recovered to the control levels at 7 days after AMI. In addition, there was a correlation between circulating miR-133 or miR-328 levels and cardiac troponin I. Furthermore, circulating miR-133 or miR-328 showed no significant changes in AMI patients with tachyarrhythmia (n=24) or bradyarrhythmia (n=26) compared to those in patients without arrhythmias. Receiver operating characteristic curve analysis revealed that the areas under the curve of miR-133 or miR-328 in plasma and whole blood were 0.890, 0.702 and 0.810, 0.872, respectively (all p<0.05). Conclusion The miR-133 and miR-328 levels in plasma and whole blood in AMI patients were increased compared to those in control subjects. These miRNAs may represent novel biomarkers of AMI.

ATP-sensitive potassium channels (KATP) are involved in a diverse array of physiologic functions including protection of tissue against ischemic insult, regulation of vascular tone, and modulation of insulin secretion. To improve our understanding of the role of K ATP in these processes, we used a gene-targeting strategy to generate mice with a disruption in the muscle-specific K ATP regulatory subunit, SUR2. Insertional mutagenesis of the Sur2 locus generated homozygous null (Sur2 ؊/؊ ) mice and heterozygote (Sur2 ؉/؊ ) mice that are viable and phenotypically similar to their wild-type littermates to 6 weeks of age despite, respectively, half or no SUR2 mRNA expression or channel activity in skeletal muscle or heart. Sur2 ؊/؊ animals had lower fasting and fed serum glucose, exhibited improved glucose tolerance during a glucose tolerance test, and demonstrated a more rapid and severe hypoglycemia after administration of insulin. Enhanced glucose use was also observed during in vivo hyperinsulinemic euglycemic clamp studies during which Sur2 ؊/؊ mice required a greater glucose infusion rate to maintain a target blood glucose level. Enhanced insulin action was intrinsic to the skeletal muscle, as in vitro insulin-stimulated glucose transport was 1.5-fold greater in Sur2 ؊/؊ muscle than in wild type. Thus, membrane excitability and K ATP activity, to our knowledge, seem to be new components of the insulin-stimulated glucose uptake mechanism, suggesting possible future therapeutic approaches for individuals suffering from diabetes mellitus.

Previously, we have shown abnormalities in Vmax and in the recovery of Vmax in myocytes dispersed from the epicardial border zone (EBZ) of the 5-day infarcted canine heart (myocytes from the EBZ [IZs]). Thus, we sought to determine the characteristics of the whole-cell Na+ current (INa) in IsZs and compare them with the INa of cells from noninfarcted hearts (myocytes from noninfarcted epicardium [NZs]). INa was recorded using patch-clamp techniques under conditions that eliminated contaminating currents and controlled INa for measurement (19 degrees C, 5 mmol/L [Na+]zero). Peak INa density (at -25 mV) was significantly reduced in IZs (4.9 +/- 0.44 pA/pF, n = 36) versus NZs (12.8 +/- 0.55 pA/pF, n = 54; P < .001), yet the half-maximal activation voltage (V0.5), time course of decay, and time to peak INa were no different. However, in IZs, V0.5 of the availability curve (I/Imax curve) was shifted significantly in the hyperpolarizing direction (-80.2 +/- 0.48 mV in NZs [n = 45] versus -83.9 +/- 0.59 mV in IZs [n = 27], P < .01). Inactivation of INa directly from a depolarized prepotential (-60 mV) was significantly accelerated in IZs versus NZs (fast and slow time constants [T1 and T2, respectively] were as follows: NZs [n = 28], T1 = 71.5 +/- 5.6 ms and T2 = 243.7 +/- 17.1 ms; IZs [n = 21], T1 = 36.3 +/- 2.4 ms and T2 = 153 +/- 11.3 ms; P < .001). Recovery of INa from inactivation was dependent on the holding potential (VH) in both cell types but was significantly slower in IZs. At (VH) = -90 mV, INa recovery had a lag in 18 (82%) of 22 IZs (with a 17.6 +/- 1.5-ms lag) versus 2 (9%) of 22 NZs (with 5.9- and 8.7-ms lags); at VH = -100 mV, T1 = 60.9 +/- 2.6 ms and T2 = 352.8 +/- 28.1 ms in NZs (n = 41) versus T1 = 76.3 +/- 4.8 ms and T2 = 464.4 +/- 47.2 ms in IZs (n = 26) (P < .002 and P < .03, respectively); at VH = -110 mV, T1 = 33.4 +/- 1.8 ms and T2 = 293.5 +/- 33.6 ms in NZs (n = 21) versus T1 = 44.3 +/- 2.9 ms and T2 = 388.4 +/- 38 ms in IZs (n = 18) (P < .002 and P < .07, respectively). In sum, INa is reduced, and its kinetics are altered in IZs. These changes may underlie the altered excitability and postrepolarization refractoriness of the ventricular fibers of the EBZ, thus contributing to reentrant arrhythmias in the infarcted heart.

IntroductionK ATP channels respond to changes in the intracellular ATP content by altering a cell's membrane potential (1, 2). K ATP channels are widely expressed in neural, endocrine, and muscle tissues where they are inhibited by ATP and stimulated by ADP. K ATP channels are an octameric complex consisting of four potassium channel subunits, either Kir6.1 or Kir6.2, and four sulfonylurea receptor subunits, SUR1 or SUR2 (3-9). SURs, named for their ability to bind with high affinity the hypoglycemic sulfonylurea agents, are members of the ATP-binding cassette (ABC) transporter family of transmembrane proteins. SUR1 and SUR2 are 70% identical proteins encoded by different genes. SUR2 undergoes differential splicing altering its carboxy-terminal 42 amino acids, yielding channels with unique pharmacologic properties.In cardiomyocytes, where K ATP channels modulate protection from ischemia, SUR1 and SUR2 are coexpressed (10, 11). In smooth muscle and voluntary striated muscle, only SUR2 is expressed (11). The physiology and pharmacology of K ATP channels have been most extensively studied in the pancreatic β cell. Here, sulfonylurea agents such as glibenclamide inhibit K ATP channels by binding to SUR1, which results in the closure of the channel and the stimulation of insulin release (3). In vascular smooth muscle, potassium channel openers, such as nicorandil, implicate K ATP channels in the regulation of tonic vasomotor activity. These agents, useful in the treatment of hypertension and angina, open K ATP channels leading to potassium efflux, membrane hyperpolarization, and vasodilation (12-14). Potassium channel openers alter membrane potential through K ATP channels and thereby activate voltage-dependent calcium channels producing changes in vascular smooth muscle contractility (15). In skeletal muscle, K ATP channels affect glucose metabolism. Using mice with a targeted disruption of the Sur2 gene (16), we demonstrated that the loss of K ATP channels increased insulin responsiveness mediated by striated muscle.The diversity of responses to individual pharmacologic agents that act through K ATP channels derives in part from the tissue-specific expression of K ATP subunits and the composition of K ATP channels within a cell. Since pharmacologic agents act through the SUR subunit, the significant homology between SUR isoforms and their overlapping expression pattern complicates the interpretation of pharmacologic studies in This article was published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.