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Introduction: SARS-CoV-2 has been found in the heart of COVID-19 patients. It is unclear how the virus passes from the upper respiratory tract to the myocardium. Hypothesis: SARS-CoV-2 is present in the blood of COVID-19 infected patients. Methods: We targeted two viropotins, Orf3a and E, in SARS-CoV-2. Orf3a and E form non-voltage-gated ion channels. A combined fluorescence potassium ion assay with three channel modulators (4-aminopyridine, emodin-Orf3a channel blocker, gliclazide-E channel blocker) was developed to detect SARS-CoV-2 Orf3a/E channel activity. In blood samples, we subtracted the fluorescence signals in the absence and presence of emodin/gliclazide to detect Orf3a and E channel activity. Results: In lentivirus spiked samples, we detected significant channel activity of Orf3a/E based on increase in fluorescence induced by 4-aminopyridine, and this increase in fluorescence was inhibited by emodin and gliclazide. In 18 antigen/PCR positive samples, our test results found 15 are positive, demonstrating 83.3% concordance. In 24 antigen/PCR negative samples, our test results found 21 are negative, showing 87.5% concordance. Conclusions: We developed a cell-free, rapid, and high-throughput test that can detect Orf3a/E channel activity of SARS-CoV-2 in blood samples from COVID-19 infected individuals, which provides a likely explanation that the virus spreads to the heart via blood circulation.

Our previous results have demonstrated that tyrosine kinase inhibition reduces i(f) in rabbit SA node myocytes, suggesting that tyrosine kinases regulate i(f). One receptor tyrosine kinase the EGF receptor kinase is known to increase heart rate. To determine if this action is mediated through changes in i(f), we examined the effect of epidermal growth factor (EGF) on i(f) with the permeabilized patch-clamp technique. 0.1 microM EGF increased i(f) amplitude in response to single-step hyperpolarizations in the diastolic range of potentials. This increase was 20+/-3%, n=11 at -75 mV. This effect is caused by activating a tyrosine kinase because 50 microM genistein, a tyrosine kinase inhibitor, eliminated this EGF action. A two-step pulse protocol showed that maximal i(f) conductance was increased by EGF. We further examined this conductance change by constructing the activation curve. The maximal i(f) conductance was increased by 23% with no change in midpoint, V(1/2), control=-74+/-2 mV, V(1/2) EGF=-74+/-1 mV. Thus EGF acts via a tyrosine kinase to increase maximal i(f) conductance with no change in the voltage dependence of activation. These results suggest that EGF effects on i(f) contribute to the positive chronotropic effect of EGF on SA node.

Background The classic calciotropic hormone parathyroid hormone (PTH) and its paracrine factor parathyroid hormone–related protein (PTHrP) both increase heart rate. Methods and Results We used standard electrophysiological techniques to study the effects of PTH and PTHrP on isolated rabbit sinus node, isolated canine Purkinje fibers, and disaggregated rabbit sinus node myocytes. Sinus node maximum diastolic potential, activation voltage, and amplitude were unchanged by PTH or PTHrP ( P >.05). However, the slope of phase 4 and the automatic rate were increased at PTH and PTHrP ≥10 nmol/L ( P I f pacemaker current in sinus node. PTH 12.5 nmol/L and PTHrP 12.5 to 18 nmol/L increased I f at −65 mV by 68±41% (n=5) and 69±50% (n=5), respectively. Actions of both agents were reversible. The increase in I f appeared to result from a change in maximal conductance and not a shift in the voltage dependence of activation. Conclusions These observations provide, for the first time, direct electrophysiological support for the chronotropic actions of PTH and PTHrP. They suggest that classic hormones and paracrine factors can have multiple functions and that in the case of PTH and PTHrP, a newly recognized action is to alter automaticity directly.

We have investigated the actions of vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) on the pacemaker current (I(f)) in canine Purkinje fibers. On voltage pulses to the middle of the I(f) activation range, VIP reversibly increases I(f), whereas NPY reversibly decreases I(f). A three-pulse voltage protocol suggests that VIP shifts I(f) activation in the positive direction and that NPY shifts I(f) activation in the negative direction on the voltage axis without changing maximal I(f) conductance. These effects of VIP and NPY on I(f) are exerted through their specific peptide receptors, since the effects are blocked by VIP and NPY receptor antagonists. VIP and NPY are colocalized in cardiac parasympathetic and sympathetic nerve endings, respectively, and can be released preferentially on high and long-lasting nerve stimulation. Given this colocalization and frequency-dependent release, these results suggest a role for these neuropeptides in controlling cardiac I(f) and consequently heart rate.

Myocytes were isolated from newborn and adult rat ventricle. Using the whole-cell patch clamp, the two cell populations were compared for the presence of the hyperpolarization-activated pacemaker current if. As in other mammalian species, the threshold voltage in acutely dissociated adult rat myocytes was extremely negative (-113 +/- 5 mV; n=12). In contrast, threshold in newborn cells was relatively positive, regardless of whether measured in acutely dissociated (-72 +/- 2 mV; n=6) or cultured cells (-70 +/- 2 mV; n=9). Current density was not reduced in the adult. These results suggest that with development the ventricle assumes its non-pacemaker function, at least in part, by a shift of the voltage dependence of if outside the physiological range.

The actions of the phosphatase inhibitor calyculin A on the pacemaker current i(f) were studied in canine Purkinje fibers and myocytes. Calyculin A increased i(f) in response to hyperpolarizations toward the middle of the i(f) activation curve. A three pulse protocol indicated this increase was due to a positive shift of i(f) activation on the voltage axis. Taken together with our previous results (that kinase inhibition with H7 shifts i(f) activation in the negative direction on the voltage axis (2)), these results suggest that phosphorylation is an important regulator of the voltage dependence of i(f) activation.

Abstract —The HCN family of ion channel subunits underlies the currents I f in heart and I h and I q in the nervous system. In the present study, we demonstrate that minK-related peptide 1 (MiRP1) is a β subunit for the HCN family. As such, it enhances protein and current expression as well as accelerating the kinetics of activation. Because MiRP1 also functions as a β subunit for the cardiac delayed rectifier I Kr , these results suggest that this peptide may have the unique role of regulating both the inward and outward channels that underlie cardiac pacemaker activity. The full text of this article is available at http://www.circresaha.org.

Abstract —The Ca 2+ -independent portion of transient outward K + current ( I to ) exhibits a transmural gradient in ventricle. To investigate control mechanisms for this gradient, we studied canine epicardial and endocardial ventricular myocytes with use of the whole-cell patch-clamp technique. I to was larger in amplitude, had a more negative voltage threshold for activation, and had a more negative midpoint of inactivation in epicardium. Recovery from inactivation was >10-fold slower in endocardium. Incubation of epicardial myocytes with angiotensin II for 2 to 52 hours altered I to to resemble unincubated endocardium and reduced the amplitude of the phase 1 notch of the action potential. In contrast, incubation of endocardial myocytes with losartan for 2 to 52 hours altered I to to resemble unincubated epicardium and induced a phase 1 notch in the action potential. With RNase protection assays, we determined that incubations with angiotensin II or losartan did not alter mRNA levels for either Kv4.3 or Kv1.4; thus, a change in the α subunit for I to is unlikely to be responsible. To test whether posttranslational modification produced the effects of angiotensin II, we coexpressed Kv4.3 and the angiotensin II type 1a receptor in Xenopus oocytes. Incubation with angiotensin II increased the time constant for recovery from inactivation of the expressed current by 2-fold with an incubation time constant of 3.7 hours. No effect on activation or inactivation voltage dependence was observed. These results demonstrate that the properties of I to in endocardium and epicardium are plastic and likely under the tonic-differing influence of the renin-angiotensin system.

Abstract —HCN cation channel mRNA expression was determined in the rabbit heart and neonatal and adult rat ventricle using RNase protection assays. In the rabbit SA node, the dominant HCN transcript is HCN4, representing >81% of the total HCN message. HCN1 is also expressed, representing >18% of the total HCN mRNA. Rabbit Purkinje fibers contained almost equal amounts of HCN1 and HCN4 transcripts with low levels of HCN2, whereas rabbit ventricle contained predominantly HCN2. The SA node contained 25 times the total HCN message of Purkinje fibers and 140 times the total HCN message of ventricle. No reports of hyperpolarization-activated current ( I f ) exist in rabbit Purkinje fibers, and we could not record I f in rabbit ventricular myocytes. To investigate the possible role of isoform switching in determining the voltage dependence of I f , we determined the prevalence of HCN isoforms in neonatal and adult rat ventricle. We had previously determined the threshold for activation of I f to be ≈−70 mV in neonatal rat ventricle and −113 mV in adult rat ventricle. In both neonatal and adult rat ventricle, only HCN2 and HCN4 transcripts are present. The ratio of HCN2 to HCN4 is ≈5:1 in the neonate and 13:1 in the adult. Taken together, these results suggest that different cardiac regions express different isoforms of the HCN family. The HCN1 and HCN4 isoforms are most closely associated with a depolarized threshold for I f activation, whereas the HCN2 isoform is associated with a more negative activation curve. The full text of this article is available at http://www.circresaha.org.

Background —Cardiac memory refers to an altered T-wave morphology induced by ventricular pacing or arrhythmias that persist for variable intervals after resumption of sinus rhythm. Methods and Results —We induced long-term cardiac memory (LTM) in conscious dogs by pacing the ventricles at 120 bpm for 3 weeks. ECGs were recorded daily for 1 hour, during which time pacing was discontinued. At terminal study, the heart was removed and the electrophysiology of left ventricular epicardial myocytes was investigated. Control (C) and LTM ECG did not differ, except for T-wave amplitude, which decreased from 0.12±0.18 to −0.34±0.21 mV (±SEM, P P P I to ) was investigated by whole-cell patch clamp. No difference in capacitance was seen in C and LTM myocytes. I to activated on membrane depolarization to −25±1 mV in C and −7±1 mV ( P I to density was reduced in LTM myocytes, and a decreased mRNA level for Kv4.3 was observed. Recovery of I to from inactivation was significantly prolonged: it was 531±80 ms (n=10) in LTM and 27±6 ms (n=9) in C ( P Conclusions — I to changes are associated with and can provide at least a partial explanation for action-potential and T-wave changes occurring with LTM.

The expression of 15 different K + channels in canine heart was examined, and a new K + channel gene (Kv4.3), which encodes a rapidly inactivating K + current, is described. The Kv4.3 channel was found to have biophysical and pharmacological properties similar to the native canine transient outward current (I to ). The Kv4.3 gene is also expressed in human and rat heart. It is concluded that the Kv4.3 channel underlies the bulk of the I to in canine ventricular myocytes, and probably in human myocytes. Both the Kv4.3 and Kv4.2 channels are likely to contribute to the I to in rat heart, and differential expression of these two channels can account for observed differences in the kinetic properties of the I to in different regions of rat ventricle. There are significant differences in the pattern of K + channel expression in canine heart, compared with rat heart, and these differences may be an adaptation to the different requirements for cardiac function in mammals of markedly different sizes. It is possible that the much longer ventricular action potential duration observed in canine heart compared with rat heart is due, in part, to the lower levels of Kv1.2, Kv2.1, and Kv4.2 gene expression in canine heart.

1. Single cells enzymatically isolated from canine ventricle and canine Purkinje fibres were studied with the whole-cell patch clamp technique, and the properties of the pacemaker current i(f) compared. 2. Steady-state i(f) activation occurred in canine ventricular myocytes at more negative potentials (-120 to -170 mV) than in canine Purkinje cells (-80 to -130 mV). 3. Reversal potentials were obtained in various extracellular Na+ (140, 79 or 37 mM) and K+ concentrations (25, 9 or 5.4 mM) to determine the ionic selectivity of i(f) in the ventricle. The results suggest that this current was carried by both sodium and potassium ions. 4. The plots of the time constants of i(f) activation against voltage were 'bell shaped' in both canine ventricular and Purkinje myocytes. The curve for the ventricular myocytes was shifted about 30 mV in the negative direction. In both ventricular and Purkinje myocytes, the fully activated I-V relationship exhibited outward rectification in 5.4 mM extracellular K+. 5. Calyculin A (0.5 microM) increased i(f) by shifting its activation to more positive potentials in ventricular myocytes. Protein kinase inhibition by H-7 (200 microM) or H-8 (100 microM) reversed the positive voltage shift of i(f) activation. This effect of calyculin A also occurred when the permeabilized patch was used for whole-cell recording. 6. These results indicate i(f) is present in ventricular myocytes. If shifted to more positive potentials i(f) could play a role in ischaemia-induced ventricular arrhythmias. The negative shift of i(f) in the ventricle might play a role in differentiating non-pacing regions of the heart from those regions that pace.