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We previously demonstrated that a two-cell syncytium, composed of a ventricular myocyte and an mHCN2 expressing cell, recapitulated most properties of in vivo biological pacing induced by mHCN2-transfected hMSCs in the canine ventricle. Here, we use the two-cell syncytium, employing dynamic clamp, to study the roles of gf (pacemaker conductance), gK1 (background K+ conductance), and gj (intercellular coupling conductance) in biological pacing. We studied gf and gK1 in single HEK293 cells expressing cardiac sodium current channel Nav1.5 (SCN5A). At fixed gf, increasing gK1 hyperpolarized the cell and initiated pacing. As gK1 increased, rate increased, then decreased, finally ceasing at membrane potentials near EK. At fixed gK1, increasing gf depolarized the cell and initiated pacing. With increasing gf, rate increased reaching a plateau, then decreased, ceasing at a depolarized membrane potential. We studied gj via virtual coupling with two non-adjacent cells, a driver (HEK293 cell) in which gK1 and gf were injected without SCN5A and a follower (HEK293 cell), expressing SCN5A. At the chosen values of gK1 and gf oscillations initiated in the driver, when gj was increased synchronized pacing began, which then decreased by about 35% as gj approached 20 nS. Virtual uncoupling yielded similar insights into gj. We also studied subthreshold oscillations in physically and virtually coupled cells. When coupling was insufficient to induce pacing, passive spread of the oscillations occurred in the follower. These results show a non-monotonic relationship between gK1, gf, gj, and pacing. Further, oscillations can be generated by gK1 and gf in the absence of SCN5A.

BACKGROUND This study compared aortic pressures estimated using a SphygmoCor XCEL PWA device (ATCOR, Naperville, IL) noninvasively with aortic pressures obtained using pressure catheters during catheterization procedures and analyzed the impact of a linear-fit function on the estimated pressure values. METHODS One hundred and thirty-six patients scheduled for cardiac catheterization procedure were enrolled in IRB approved studies. Catheterization procedures were performed according to standard-of-care to acquire aortic pressure measurements. Immediately after the catheterization procedure with the pressure catheters removed, while the patients were still in the catheterization laboratory, central aortic pressures were estimated with the SphygmoCor device (using its inbuilt transfer function). The error between measured and estimated aortic pressures was evaluated using Bland–Altman analysis (n = 93). A linear-fit was performed between the measured and estimated pressures, and using the linear equation the error measurements were repeated. A bootstrap analysis was performed to test the generalizability of the linear-fit function. In a subset of cases (n = 13), central aortic pressure values were also obtained using solid-state high-fidelity catheters (Millar, Houston, TX), and the error measurements were repeated. RESULTS The magnitude of errors between the measured and estimated aortic pressures (mean errors >6.4 mm Hg; mean errors >8.0 mm Hg in the subset) were reduced to less than 1 mm Hg after using the linear-fit function derived in this study. CONCLUSIONS For the population examined in this study, the SphygmoCor data must be used with the linear-fit function to obtain aortic pressures that are comparable to the measurements obtained using pressure catheters. CLINICAL TRIALS REGISTRATION Trial Numbers NCT03243942 and NCT03245255.

One promising approach to cancer therapeutics is to induce changes in gene expression that either reduce cancer cell proliferation or induce cancer cell death. Therefore, delivering oligonucleotides (siRNA/miRNA) that target specific genes or gene programs might have a potential therapeutic benefit. The aim of this study was to examine the potential of cell-based delivery of oligonucleotides to cancer cells via two naturally occurring intercellular pathways: gap junctions and vesicular/exosomal traffic. We utilized human mesenchymal stem cells (hMSCs) as delivery cells and chose to deliver in vitro two synthetic oligonucleotides, AllStars HS Cell Death siRNA and miR-16 mimic, as toxic (therapeutic) oligonucleotides targeting three cancer cell lines: prostate (PC3), pancreatic (PANC1) and cervical (HeLa). Both oligonucleotides dramatically reduced cell proliferation and/or induced cell death when transfected directly into target cells and delivery hMSCs. The delivery and target cells we chose express gap junction connexin 43 (Cx43) endogenously (PC3, PANC1, hMSC) or via stable transfection (HeLaCx43). Co-culture of hMSCs (transfected with either toxic oligonucleotide) with any of Cx43 expressing cancer cells induced target cell death (~20% surviving) or senescence (~85% proliferation reduction) over 96 hours. We eliminated gap junction-mediated delivery by using connexin deficient HeLaWT cells or knocking out endogenous Cx43 in PANC1 and PC3 cells via CRISPR/Cas9. Subsequently, all Cx43 deficient target cells co-cultured with the same toxic oligonucleotide loaded hMSCs proliferated, albeit at significantly slower rates, with cell number increasing on average ~2.2-fold (30% of control cells) over 96 hours. Our results show that both gap junction and vesicular/exosomal intercellular delivery pathways from hMSCs to target cancer cells deliver oligonucleotides and function to either induce cell death or significantly reduce their proliferation. Thus, hMSC-based cellular delivery is an effective method of delivering synthetic oligonucleotides that can significantly reduce tumor cell growth and should be further investigated as a possible approach to cancer therapy.

Cardiac arrhythmias are the most common cause of sudden cardiac death worldwide. Lengthening the ventricular action potential duration (APD) either congenitally or via pathologic or pharmacologic means, predisposes to a life-threatening ventricular arrhythmia, Torsade de Pointes. IKs, a slowly activating K+ current plays a role in action potential repolarization. In this study, we screened a chemical library in silico by docking compounds to the voltage sensing domain (VSD) of the IKs channel. Here we show that C28 specifically shifted IKs VSD activation in ventricle to more negative voltages and reversed drug-induced lengthening of APD. At the same dosage, C28 did not cause significant changes of the normal APD in either ventricle or atrium. This study provides evidence in support of a computational prediction of IKs VSD activation as a potential therapeutic approach for all forms of APD prolongation. This outcome could expand the therapeutic efficacy of a myriad of currently approved drugs that may trigger arrhythmias.Significance statementC28, identified by in silico screening, specifically facilitated voltage dependent activation of a cardiac potassium ion channel, IKs. C28 reversed drug-induced prolongation of action potentials, but minimally affected the normal action potential at the same dosage. This outcome supports a computational prediction of modulating IKs activation as a potential therapy for all forms of action potential prolongation, and could expand therapeutic efficacy of many currently approved drugs that may trigger arrhythmias.

Introduction: Subharmonic aided pressure estimation (SHAPE) utilizes ambient pressure sensitivity of subharmonic signals from ultrasound contrast agents. The objective was to evaluate SHAPE with Definity (Lantheus Medical Imaging, Inc.) and Sonazoid (GE Healthcare) microbubbles for intra-cardiac pressure estimation. Hypothesis: Errors between SHAPE and pressures obtained during cardiac catheterization ≤ 5 mmHg. Methods: IRB approved this study. Consenting patients received an infusion of Definity (56 patients; 2 vials in 50 mL of saline; infusion rate: 4-10 mL/min) or Sonazoid (77 patients; rate (mL/hour) = 0.18 x weight in kg co-infused with saline at 120 mL/hour) during cardiac catheterization. Subharmonic data was acquired using a SonixTablet (PA 4/2 array; BK Ultrasound) synchronously with pressures from the left and right ventricles and the aorta (for left heart catheterizations only). Subharmonic data (in dB) was converted to pressure values (in mmHg) using calibration factors (mmHg/dB) based on data obtained from the aorta - utilizing pressures from the aorta either during the catheterization procedure or pressures obtained using a SphygmoCor (AtCor Medical Pty. Ltd.) device after the catheterization procedure. Clinically relevant pressures between the SHAPE technique and the pressure catheter were compared. Results: Correlation coefficient between the subharmonic and pressure data was -0.8 ± 0.1. With Definity, mean absolute errors ranged from 2.9 ± 1.5 to 5.0 ± 4.2 mmHg and from 4.4 ± 5.7 to 23.7 ± 28.3 mmHg for calibration factors utilizing aortic pressures from pressure catheter and SphygmoCor, respectively. For Sonazoid microbubbles, these errors ranged from 7.9 ± 12.0 to 10.1 ± 12.4 mmHg and from 7.2 ± 11.5 to 15.0 ± 23.2 mmHg, respectively. Two adverse events occurred during Definity infusion; these were resolved with return to baseline conditions. Conclusion: SHAPE may be useful for estimating intra-cardiac pressures noninvasively.

The objective of this work was to investigate if central cardiac pressures are obtainable noninvasively using the subharmonic aided pressure estimation (SHAPE) technique with Definity (Lantheus Medical Imaging Inc, N Billerica, MA, USA) or Sonazoid (GE Healthcare, Oslo, Norway) microbubbles. Patients scheduled for a left and/or right heart catheterization procedure and providing written informed consent were included in IRB approved clinical trials (NCT03243942 for Definity; NCT03245255 for Sonazoid). A standard-of-care catheterization procedure was performed advancing clinically used pressure catheter into the left and/or right ventricles and/or the aorta. After pressure catheter placement, patients received an infusion of either Definity (56 patients; 2 vials in 50 mL of saline; infusion rate: 4–10 mL/min) or Sonazoid (60 patients; rate (mL/hour) = 0.18 mL/hour/kg x weight in kg co-infused with saline at 120 mL/hour). A customized interface on a SonixTablet scanner (BK Ultrasound, Peabody, MA, USA) was used to acquire SHAPE data synchronously with the pressure catheter data. Linear correlation between the SHAPE and pressure catheter data were computed using MATLAB (Mathworks, Natick, MA, USA). Central aortic pressures were estimated using cuff-based brachial pressure measurements with a SphygmoCor device (AtCor Medical Pty Ltd, West Ryde, NSW, Australia). Central aortic pressures and SHAPE data from the aorta were used to calculate a conversion factor (in mmHg/dB) for each patient to estimate pressures and determine errors associated with the SHAPE technique. Two adverse events were observed during Definity infusion; both were resolved. Errors between the pressure catheter and SHAPE derived mean diastolic pressures were less than 5 mmHg when using Definity microbubbles and greater than 5 mmHg when using Sonazoid microbubbles. These results indicate that SHAPE is a useful technique to noninvasively obtain central cardiac pressures.

We previously demonstrated that a two-cell syncytium, composed of a ventricular myocyte and an mHCN2 expressing cell, recapitulated most properties of in vivo biological pacing induced by mHCN2-transfected hMSCs in the canine ventricle. Here, we use the two-cell syncytium, employing dynamic clamp, to study the roles of g

KCNQ family K+ channels (KCNQ1-5) in the heart, nerve, epithelium and ear require phosphatidylinositol 4,5-bisphosphate (PIP2) for voltage dependent activation. While membrane lipids are known to regulate voltage sensor domain (VSD) activation and pore opening in voltage dependent gating, PIP2 was found to interact with KCNQ1 and mediate VSD-pore coupling. Here, we show that a compound CP1, identified in silico based on the structures of both KCNQ1 and PIP2, can substitute for PIP2 to mediate VSD-pore coupling. Both PIP2 and CP1 interact with residues amongst a cluster of amino acids critical for VSD-pore coupling. CP1 alters KCNQ channel function due to different interactions with KCNQ compared with PIP2. We also found that CP1 returned drug-induced action potential prolongation in ventricular myocytes to normal durations. These results reveal the structural basis of PIP2 regulation of KCNQ channels and indicate a potential approach for the development of anti-arrhythmic therapy., Yongfeng Liu, Xianjin Xu, Junyuan Gao et al. perform in silico screening targeting compounds that resemble both KCNQ1 and PIP2 structures and identify CP1 as a compound that could restore KCNQ1 currents after PIP2 depletion. Like PIP2, this compound can mediate VSD-pore coupling of the channel, albeit via a different mechanism from PIP2.

This review focuses on the biophysical properties and structure of the pore and vestibule of homotypic gap junction channels as they relate to channel permeability and selectivity. Gap junction channels are unique in their sole role to connect the cytoplasm of two adjacent cells. In general, these channels are considered to be poorly selective, possess open probabilities approximating unity, and exhibit mean open times ranging from milliseconds to seconds. These properties suggest that such channels can function as delivery pathways from cell to cell for solutes that are significantly larger than monovalent ions. We have taken quantitative data from published works concerning unitary conductance, ion flux, and permeability for homotypic connexin 43 (Cx43), Cx40, Cx26, Cx50, and Cx37, and performed a comparative analysis of conductance and/or ion/solute flux versus diffusion coefficient. The analysis of monovalent cation flux portrays the pore as equivalent to an aqueous space where hydrogen bonding and weak interactions with binding sites dominate. For larger solutes, size, shape and charge are also significant components in determining the permeation rate. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

This study evaluated the feasibility of noninvasive intracardiac pressure estimation using subharmonic signals from ultrasound contrast agents in humans. This IRB approved proof-of-concept study included 15 consenting patients scheduled for left and right heart catheterization. During the catheterization procedure, Definity (Lantheus Medical Imaging, N Billerica, MA) was infused intravenously at 4–10 ml/min. Ultrasound scanning was performed with a Sonix RP (BK Ultrasound, Richmond, Canada) using pulse inversion, three incident acoustic output levels, and 2.5 MHz transmit frequency. Radiofrequency data were processed and subharmonic amplitudes were compared with the pressure catheter data. Correlation coefficient between subharmonic signals and pressure catheter data ranged from −0.3 to −0.9. For acquisitions with optimum acoustic output, pressure errors between the subharmonic technique and catheter were as low as 2.6 mmHg. However, automatically determining optimum acoustic output during scanning for each patient remains to be addressed before clinical applicability can be decided.

While it is well known that mutation of several different ion channels can cause congenital long QT syndrome, block of IKr is widely thought to be responsible for most cases of drug-induced acquired long QT syndrome (aLQTS). In this article, we review evidence supporting another cause of aLQTS due to inhibition of phosphoinositide 3-kinase (PI3K) signaling. Inhibition of PI3K affects multiple plateau currents, reducing IKr, IKs, and ICaL while increasing the persistent sodium current (INaP). The effects of PI3K inhibitors develop slowly, requiring hours to days to reach steady state. Dofetilide and terfenadine, an antihistamine on which much of the original IKr hypothesis was based, are among the many drugs that inhibit the PI3K pathway. Reduced PI3K signaling may also play a role in aLQTS associated with diabetes. Drug safety testing to identify aLQTS risk may be improved by examining PI3K-dependent effects that develop over time.

Subharmonic aided pressure estimation (SHAPE) measures ambient pressure dependent subharmonic signals from contrast microbubbles. This study evaluated if Definity® (Lantheus Medical Imaging, N Billerica, MA, USA) or Sonazoid (GE Healthcare, Oslo, Norway) microbubbles are useful for real-time noninvasive intra-cardiac pressure estimation using SHAPE. Patients scheduled for a right and/or left heart catheterization procedure were approached to participate in this IRB approved study and provided written consent. During the standard-of-care catheterization procedure, a pressure catheter was advanced into the cardiac chambers or the aorta to obtain clinically relevant systolic and diastolic pressures. After catheter placement, the patients received either an infusion of Definity (40 patients; 2 vials in 50 mL of saline; infusion rate: 4-10 mL/min) or Sonazoid (41 patients; rate (mL/hour) = 0.18 x weight in kg co-infused with saline at 120 mL/hour). A customized interface on a SonixTablet scanner (Analogic Corporation, Peabody, MA, USA) was used to acquire SHAPE data synchronously with the pressure catheter data. Previously determined optimal transmit parameters were used for data acquisition with Definity (f transmit : 3.0 MHz; chirp down pulse) and Sonazoid (f transmit : 2.5 MHz; square wave pulse) microbubbles. An algorithm to determine optimum incident acoustic output was initiated for each patient (to account for attenuation differences). Then, SHAPE data were acquired at the optimum incident acoustic output. Linear correlation between the SHAPE and pressure catheter data were computed using MATLAB (Mathworks, Natick, MA, USA). The correlation coefficient between SHAPE and pressure catheter data obtained using Definity was −0.86 ± 0.06 (n=15) and −0.87 ± 0.06 (n=18) for data obtained from the left and right ventricles, respectively. In two cases, adverse events were observed post Definity infusion. Both events were resolved with return to baseline conditions. Using Sonazoid, the correlation coefficient between the SHAPE and pressure catheter data was −0.83± 0.05 (n=10) and −0.82 ± 0.06 (n=7) for data obtained from left and right ventricles, respectively. These results indicate that SHAPE is a useful technique to noninvasively obtain intra-cardiac pressures in real-time.

Heart rate in physiological conditions is set by the sinoatrial node (SN), the primary cardiac pacing tissue. Phosphoinositide 3-kinase (PI3K) signaling is a major regulatory pathway in all normal cells, and its dysregulation is prominent in diabetes, cancer, and heart failure. Here, we show that inhibition of PI3K slows the pacing rate of the SN in situ and in vitro and reduces the early slope of diastolic depolarization. Furthermore, inhibition of PI3K causes a negative shift in the voltage dependence of activation of the pacemaker current, IF, while addition of its second messenger, phosphatidylinositol 3,4,5-trisphosphate, induces a positive shift. These shifts in the activation of IF are independent of, and larger than, those induced by the autonomic nervous system. These results suggest that PI3K is an important regulator of heart rate, and perturbations in this signaling pathway may contribute to the development of arrhythmias.

Stem cell therapy requires a nontoxic and high-throughput method to achieve a pure cell population to prevent teratomas that can occur if even one cell in the implant has not been transformed. A promising method to detect and separate cells expressing a particular gene is RNA beacon technology. However, developing a successful, specific beacon to a particular transfected gene can take months to develop and in some cases is impossible. Here, we report on an off-the-shelf universal beacon that decreases the time and cost of applying beacon technology to select any living cell population transfected with an exogenous gene. Significance Stem cell therapy requires pure cell populations. RNA beacons have a high potential to select and separate differentiated cells from undifferentiated cells. However, development of a beacon for a specific gene can be difficult and time-consuming. An RNA tag was developed that can be attached to any gene without affecting the protein product. The tagged gene is detected by an off-the-shelf RNA beacon that can be used for cell selection.

Our previous work on angiotensin II-mediated electrical-remodeling in canine left ventricle, in connection with a long history of other studies, suggested the hypothesis: increases in mechanical load induce autocrine secretion of angiotensin II (A2), which coherently regulates a coterie of membrane ion transporters in a manner that increases contractility. However, the relation between load and A2 secretion was correlative. We subsequently showed a similar or identical system was present in murine heart. To investigate whether the relation between mechanical load and A2-mediated electrical remodeling was causal, we employed transverse aortic constriction in mice to subject the left ventricle to pressure overload for short-term (1 to 2 days) or long-term (1 to 2 weeks) periods. Heart-to-body weight ratios and cell capacitance measurements were used to determine hypertrophy. Whole-cell patch clamp recordings of the predominant repolarization currents Ito,fast and IK,slow were used to assess electrical remodeling. Hearts or myocytes subjected to long-term load displayed significant hypertrophy, which was not evident in short-term load. However, short-term load induced significant reductions in Ito,fast and IK,slow. Incubation of these myocytes with the angiotensin II type 1 receptor inhibitor saralasin for 2 hours restored Ito,fast and IK,slow to control levels. The number of Ito.fast or IK,slow channels did not change with A2 or long-term load, however the hypertrophic increase in membrane area reduced the current densities for both channels. For Ito,fast but not IK,slow there was an additional reduction that was reversed by inhibition of angiotensin receptors. These results suggest increased load activates an endogenous renin angiotensin system that initially reduces Ito,fast and IK,slow prior to the onset of hypertrophic growth. However, there are functional interactions between electrical and anatomical remodeling. First, hypertrophy tends to reduce all current densities. Second, the hypertrophic program can modify signaling between the angiotensin receptor and target current.

Electronic pacemakers are the standard therapy for bradycardia-related symptoms but have shortcomings. Over the past 15 years, experimental evidence has demonstrated that gene and cell-based therapies can create a biological pacemaker. Recently, physiologically acceptable rates have been reported with an adenovirus-based approach. However, adenovirus-based protein expression does not last more than 4 weeks, which limits its clinical applicability. Cell-based platforms are potential candidates for longer expression. Currently there are two cell-based approaches being tested: (i) mesenchymal stem cells used as a suitcase for delivering pacemaker genes and (ii) pluripotent stem cells differentiated down a cardiac lineage with endogenous pacemaker activity. This review examines the current achievements in engineering a biological pacemaker, defines the patient population for whom this device would be useful and identifies the challenges still ahead before cell therapy can replace current electronic devices.

Diabetes is an independent risk factor for sudden cardiac death and ventricular arrhythmia complications of acute coronary syndrome. Prolongation of the QT interval on the electrocardiogram is also a risk factor for arrhythmias and sudden death, and the increased prevalence of QT prolongation is an independent risk factor for cardiovascular death in diabetic patients. The pathophysiological mechanisms responsible for this lethal complication are poorly understood. Diabetes is associated with a reduction in phosphoinositide 3-kinase (PI3K) signaling, which regulates the action potential duration (APD) of individual myocytes and thus the QT interval by altering multiple ion currents, including the persistent sodium current INaP. Here, we report a mechanism for diabetes-induced QT prolongation that involves an increase in INaP caused by defective PI3K signaling. Cardiac myocytes of mice with type 1 or type 2 diabetes exhibited an increase in APD that was reversed by expression of constitutively active PI3K or intracellular infusion of phosphatidylinositol 3,4,5-trisphosphate (PIP3), the second messenger produced by PI3K. The diabetic myocytes also showed an increase in INaP that was reversed by activated PI3K or PIP3. The increases in APD and INaP in myocytes translated into QT interval prolongation for both types of diabetic mice. The long QT interval of type 1 diabetic hearts was shortened by insulin treatment ex vivo, and this effect was blocked by a PI3K inhibitor. Treatment of both types of diabetic mouse hearts with an INaP blocker also shortened the QT interval. These results indicate that downregulation of cardiac PI3K signaling in diabetes prolongs the QT interval at least in part by causing an increase in INaP. This mechanism may explain why the diabetic population has an increased risk of life-threatening arrhythmias.

Gating of ion channels by ligands is fundamental to cellular function, and ATP serves as both an energy source and a signaling molecule that modulates ion channel and transporter functions. The slowly activating K(+) channel I(Ks) in cardiac myocytes is formed by KCNQ1 and KCNE1 subunits that conduct K(+) to repolarize the action potential. Here we show that intracellular ATP activates heterologously coexpressed KCNQ1 and KCNE1 as well as I(Ks) in cardiac myocytes by directly binding to the C terminus of KCNQ1 to allow the pore to open. The channel is most sensitive to ATP near its physiological concentration, and lowering ATP concentration in cardiac myocytes results in I(Ks) reduction and action potential prolongation. Multiple mutations that suppress I(Ks) by decreasing the ATP sensitivity of the channel are associated with the long QT (interval between the Q and T waves in electrocardiogram) syndrome that predisposes afflicted individuals to cardiac arrhythmia and sudden death. A cluster of basic and aromatic residues that may form a unique ATP binding site are identified; ATP activation of the wild-type channel and the effects of the mutations on ATP sensitivity are consistent with an allosteric mechanism. These results demonstrate the activation of an ion channel by intracellular ATP binding, and ATP-dependent gating allows I(Ks) to couple myocyte energy state to its electrophysiology in physiologic and pathologic conditions.

Background— Drugs are screened for delayed rectifier potassium current ( I Kr ) blockade to predict long QT syndrome prolongation and arrhythmogenesis. However, single-cell studies have shown that chronic (hours) exposure to some I Kr blockers (eg, dofetilide) prolongs repolarization additionally by increasing late sodium current ( I Na-L ) via inhibition of phosphoinositide 3-kinase. We hypothesized that chronic dofetilide administration to intact dogs prolongs repolarization by blocking I Kr and increasing I Na-L . Methods and Results— We continuously infused dofetilide (6–9 μg/kg bolus+6–9 μg/kg per hour IV infusion) into anesthetized dogs for 7 hours, maintaining plasma levels within the therapeutic range. In separate experiments, myocardial biopsies were taken before and during 6-hour intravenous dofetide infusion, and the level of phospho-Akt was determined. Acute and chronic dofetilide effects on action potential duration (APD) were studied in canine left ventricular subendocardial slabs using microelectrode techniques. Dofetilide monotonically increased QTc and APD throughout 6.5-hour exposure. Dofetilide infusion during ≥210 minutes inhibited Akt phosphorylation. I Na-L block with lidocaine shortened QTc and APD more at 6.5 hours than at 50 minutes (QTc) or 30 minutes (APD) dofetilide administration. In comparison, moxifloxacin, an I Kr blocker with no effects on phosphoinositide 3-kinase and I Na-L prolonged APD acutely but no additional prolongation occurred on chronic superfusion. Lidocaine shortened APD equally during acute and chronic moxifloxacin superfusion. Conclusions— Increased I Na-L contributes to chronic dofetilide effects in vivo. These data emphasize the need to include time and I Na-L in evaluating the phosphoinositide 3-kinase inhibition–derived proarrhythmic potential of drugs and provide a mechanism for benefit from lidocaine administration in clinical acquired long QT syndrome.

Biological pacing has been proposed as a physiologic counterpart to electronic pacing, and the sinoatrial node (SAN) is the general standard for biological pacemakers. We tested the expression of SAN pacemaker cell activity when implanted autologously in the right ventricle (RV). We induced complete heart block and implanted electronic pacemakers in the RV of adult mongrel dogs. Autologous SAN cells isolated enzymatically were studied by patch clamp to confirm SAN identity. SAN cells (400,000) were injected into the RV subepicardial free wall and dogs were monitored for 2 weeks. Pacemaker function was assessed by overdrive pacing and IV epinephrine challenge. SAN cells expressed a time-dependent inward current (If) activating on hyperpolarization: density = 4.3 ± 0.6 pA/pF at −105 mV. Four of the six dogs demonstrated >50% of beats originating from the implant site at 24 h. Biological pacemaker rates on days 7–14 = 45–55 bpm and post-overdrive escape times = 1.5–2.5 s. Brisk catecholamine responsiveness occurred. Dogs implanted with autologous SAN cells manifest biological pacing properties dissimilar from those of the anatomic SAN. This highlights the importance of cell and substrate interaction in generating biological pacemaker function.

Diabetes is associated with an increased risk of heart failure and the development of a cardiomyopathy whose etiology is only partially understood. Ca entry through the voltage-dependent L-type Ca channel CaV1.2 initiates the contractile cycle in cardiac myocytes. Decreased cardiac contractility and depressed CaV1.2 function have been reported in obese type 2 diabetic db/db mice. Here, we demonstrate that a reduction in phosphoinositide 3-kinase (PI3K) signaling is a major contributor to the altered function of CaV1.2 in db/db cardiac myocytes. Using the whole-cell patch clamp technique, we determined that intracellular infusion of cardiac myocytes from db/db mice with phosphatidylinositol 3,4,5-trisphosphate (PIP3), the second messenger produced by PI3K, increased the L-type Ca current (ICa,L) density nearly to the level seen in wild-type cells. PIP3 also reversed the positive shift in the voltage dependence of the steady-state current activation observed in db/db myocytes. Infusion of protein kinases that act downstream of PI3K also affected ICa,L. Akt1 and Akt2 were as effective as PIP3 in restoring the ICa,L density in db/db myocytes but did not affect the voltage dependence of current activation. The infusion of atypical PKC-ι (the human homolog of mouse PKC-λ) caused a small but significant increase in the ICa,L density and completely reversed the shift in voltage dependence of steady-state current activation. These results indicate that a defect in PI3K/PIP3/Akt/PKC-λ signaling is mainly responsible for the depressed CaV1.2 function in the hearts of db/db mice with type 2 diabetes.

Background— After the recent cloning of light-sensitive ion channels and their expression in mammalian cells, a new field, optogenetics, emerged in neuroscience, allowing for precise perturbations of neural circuits by light. However, functionality of optogenetic tools has not been fully explored outside neuroscience, and a nonviral, nonembryogenesis-based strategy for optogenetics has not been shown before. Methods and Results— We demonstrate the utility of optogenetics to cardiac muscle by a tandem cell unit (TCU) strategy, in which nonexcitable cells carry exogenous light-sensitive ion channels, and, when electrically coupled to cardiomyocytes, produce optically excitable heart tissue. A stable channelrhodopsin2 (ChR2)-expressing cell line was developed, characterized, and used as a cell delivery system. The TCU strategy was validated in vitro in cell pairs with adult canine myocytes (for a wide range of coupling strengths) and in cardiac syncytium with neonatal rat cardiomyocytes. For the first time, we combined optical excitation and optical imaging to capture light-triggered muscle contractions and high-resolution propagation maps of light-triggered electric waves, found to be quantitatively indistinguishable from electrically triggered waves. Conclusions— Our results demonstrate feasibility to control excitation and contraction in cardiac muscle by light, using the TCU approach. Optical pacing in this case uses less energy, offers superior spatiotemporal control and remote access and can serve not only as an elegant tool in arrhythmia research but may form the basis for a new generation of light-driven cardiac pacemakers and muscle actuators. The TCU strategy is extendable to (nonviral) stem cell therapy and is directly relevant to in vivo applications.

The field of biological pacing is entering its second decade of active investigation. The inception of this area of study was serendipitous, deriving largely from observations made by several teams of investigators, whose common interest was to understand the mechanisms governing cardiac impulse initiation. Research directions taken have fallen under the broad headings of gene therapy and cell therapy, and biomaterials research has also begun to enter the field. In this Review, we revisit certain milestones achieved through the construction of a 'roadmap' in biological pacing. Whether the end result will be a clinically applicable biological pacemaker is still uncertain. However, promising constructs that achieve physiologically relevant heart rates and good autonomic responsiveness are now available, and proof of principle studies are giving way to translation to large-animal models in long-term studies. Provided that interest in the field continues, the next decade should see either biological pacemakers become a clinical reality or the improvement of electronic pacemakers to a point where the biological approach is no longer a viable alternative.

High-resolution 64-MDCT images of the beating heart can be used for measurement of left atrial volume with 3D chamber reconstruction. The purpose of this study was to correlate measurements of left atrial volume obtained with clinical transthoracic echocardiography (TTE) and measurements obtained with 64-MDCT 3D reconstructions of the left atrium.Patients who underwent TTE and MDCT within 3 days were identified. TTE images were graded as excellent, good, or suboptimal. Two independent observers calculated estimates of left atrial volume from TTE and 64-MDCT images using 3D chamber reconstructions and conventional geometric assumptions on MDCT echocardiographic views.MDCT estimates of phantom volume on 3D chamber reconstructions agreed with actual volumes within 1.5%. The TTE images of 37 of the 52 patients were judged to be of good or excellent quality and were included in the analysis. Mean left atrial volume measured on 3D chamber reconstructions was 61 +/- 14 mL/m(2). Estimates of left atrial volume obtained with TTE were significantly lower (28 +/- 12 mL/m(2)) than similar estimates obtained with MDCT echocardiographic views (53 +/- 15 mL/m(2)) (p0.001). TTE left atrial volume and 3D chamber reconstruction left atrial volume exhibited moderate correlation (r = 0.60-0.70), but the correlation improved when analysis was limited to the 26 studies with excellent-quality TTE images (r = 0.71). MDCT echocardiographic estimates of left atrial volume with the area-length method had excellent correlation (r = 0.89) with and were closest to estimates made on 3D chamber reconstructions.Left atrial volume is significantly underestimated on TTE images, and TTE estimates have moderate correlation with left atrial volume measured with MDCT. Measured and estimated left atrial volumes at MDCT can provide important additive prognostic information in the care of patients undergoing MDCT for other reasons. Future studies are needed to obtain normative MDCT measurements of left atrial volume.

We examined whether coupling of a ventricular myocyte to a non-myocyte cell expressing HCN2 could create a two-cell syncytium capable of generating sustained pacing. Three non-myocyte cell types were transfected with the mHCN2 gene and used as sources of mHCN2-induced currents. They were human mesenchymal stem cells and HEK293 cells, both of which express connexin43 (Cx43), and HeLa cells transfected with Cx43. Cell–cell coupling between heterologous pairs increased with time in co-culture, and hyperpolarization of the myocyte induced HCN2 currents, indicating current transfer from the mHCN2-expressing cell to the myocyte via gap junctions. The magnitude of the HCN2 currents recorded in myocytes increased with increasing junctional conductance. Once a critical level of electrical cell–cell coupling between myocytes and mHCN2 transfected cells was exceeded spontaneous action potentials were generated at frequencies of ∼0.6 to 1.7 Hz (1.09 ± 0.05 Hz). Addition of carbenoxolone (200 μm), a gap junction channel blocker, to the media stopped spontaneous activity in heterologous cell pairs. Carbenoxolone washout restored activity. Blockade of HCN2 currents by 100 μm 9-amino-1,2,3,4-tetrahydroacridine (THA) stopped spontaneous activity and subsequent washout restored it. Neither THA nor carbenoxolone affected electrically stimulated action potentials in isolated single myocytes. In summary, the inward current evoked in the genetically engineered (HCN2-expressing) cell was delivered to the cardiac myocyte via gap junctions and generated action potentials such that the cell pair could function as a pacemaker unit. This finding lays the groundwork for understanding cell-based biological pacemakersin vivo once an understanding of delivery and target cell geometry is defined.

During the past few years, studies involving the implantation of stem cells, chemical factors, and scaffolds have demonstrated the ability to augment the mammalian heart's native regenerative capacity. Scaffolds comprised of extracellular matrix (ECM) have been used to repair myocardial defects. These scaffolds become populated with myocytes and provide regional contractile function, but quantification of the myocyte population has not yet been conducted. The purpose of this study was to quantitate the myocyte content within the ECM bioscaffold and to correlate this cell population with the regional mechanical function over time. Xenogenic ECM scaffolds derived from porcine urinary bladder were implanted into a full-thickness, surgically induced, right ventricular-free wall defect in a dog model. Zero, 2, and 8 weeks following implantation, regional function and myocyte content were determined in each patch region. Regional function did not significantly increase from 0 to 2 weeks. At 8 weeks, however, regional stroke work increased to 3.7 +/- 0.7% and systolic contraction increased to 4.4 +/- 1.2%. The myocyte content also significantly increased during that period generating a linear relationship between regional function and myocyte content. In conclusion, ECM used as a myocardial patch increases both the regional function and the myocyte content over time. The mechanical function generated in the patch region is correlated with the quantity of local tissue myocytes.

Background— Phosphoinositide 3-kinase (PI3K) p110α plays a key role in insulin action and tumorigenesis. Myocyte contraction is initiated by an inward Ca 2+ current (I Ca,L ) through the voltage-dependent L-type Ca 2+ channel (LTCC). The aim of this study was to evaluate whether p110α also controls cardiac contractility by regulating the LTCC. Methods and Results— Genetic ablation of p110 α (also known as Pik3ca ), but not p110 β (also known as Pik3cb ), in cardiac myocytes of adult mice reduced I Ca,L and blocked insulin signaling in the heart. p110α-null myocytes had a reduced number of LTCCs on the cell surface and a contractile defect that decreased cardiac function in vivo. Similarly, pharmacological inhibition of p110α decreased I Ca,L and contractility in canine myocytes. Inhibition of p110β did not reduce I Ca,L . Conclusions— PI3K p110α but not p110β regulates the LTCC in cardiac myocytes. Decreased signaling to p110α reduces the number of LTCCs on the cell surface and thus attenuates I Ca,L and contractility.

Mesenchymal stem cells (MSCs) participate in the wound healing process in mammalians. Adhesion of MSCs to endothelium is a key step in the homing of MSCs circulating in the bloodstream to the sites of injury and inflammation. Because endothelial cells (ECs) may become apoptotic under certain pro-inflammatory conditions, we investigated the effects of pro-inflammatory, TNF-alpha and IL-1 beta, and pro-apoptotic agents, actinomycin D, cycloheximide, okadaic acid, wortmannin, and staurosporine, on human MSCs (hMSCs) adhesion to ECs. Treatment of ECs with pro-apoptotic agents markedly increased adhesion of hMSCs to ECs. This adhesion correlated with reduction of mitochondrial membrane potential, inhibition of NADH dehydrogenases, and release of von Willebrand factor (vWF) by ECs. Treatment of ECs with exogenous vWF also stimulated hMSC adhesion. These data provide evidence that apoptosis of ECs may regulate homing of hMSCs to the sites of tissue injury. These results are consistent with the hypothesis that activation of apoptotic signaling pathways in ECs releases vWF which regulates hMSC adhesion to ECs.

Background— In depolarized myocardial infarct epicardial border zones, the cardiac sodium channel (SCN5A) is largely inactivated, contributing to low action potential upstroke velocity (V̇ max ), slow conduction, and reentry. We hypothesized that a fast inward current such as the skeletal muscle sodium channel (SkM1) operating more effectively at depolarized membrane potentials might restore fast conduction in epicardial border zones and be antiarrhythmic. Methods and Results— Computer simulations were done with a modified Hund-Rudy model. Canine myocardial infarcts were created by coronary ligation. Adenovirus expressing SkM1 and green fluorescent protein or green fluorescent protein alone (sham) was injected into epicardial border zones. After 5 to 7 days, dogs were studied with epicardial mapping, programmed premature stimulation in vivo, and cellular electrophysiology in vitro. Infarct size was determined, and tissues were immunostained for SkM1 and green fluorescent protein. In the computational model, modest SkM1 expression preserved fast conduction at potentials as positive as −60 mV; overexpression of SCN5A did not. In vivo epicardial border zone electrograms were broad and fragmented in shams (31.5±2.3 ms) and narrower in SkM1 (22.6±2.8 ms; P =0.03). Premature stimulation induced ventricular tachyarrhythmia/fibrillation >60 seconds in 6 of 8 shams versus 2 of 12 SkM1 ( P =0.02). Microelectrode studies of epicardial border zones from SkM1 showed membrane potentials equal to that of shams and V̇ max greater than that of shams as membrane potential depolarized ( P P =0.86). SkM1 expression in injected epicardium was confirmed immunohistochemically. Conclusions— SkM1 increases V̇ max of depolarized myocardium and reduces the incidence of inducible sustained ventricular tachyarrhythmia/fibrillation in canine infarcts. Gene therapy to normalize activation by increasing V̇ max at depolarized potentials may be a promising antiarrhythmic strategy.

The need to regenerate tissue is paramount, especially for the heart that lacks the ability to regenerate after injury. The urinary bladder extracellular matrix (ECM), when used to repair a right ventricular defect, successfully regenerated some mechanical function. The objective of the current study was to determine whether the regenerative effect of ECM could be improved by seeding the patch with human mesenchymal stem cells (hMSCs) enhanced to differentiate down a cardiac linage. hMSCs were used to form three-dimensional spheroids. The expression of cardiac proteins was determined in cells exposed to the spheroid formation and compared with nonmanipulated hMSCs. To determine whether functional calcium channels were present, the cells were patch clamped. To evaluate the ability of these cells to regenerate mechanical function, the spheroids were seeded on ECM and then implanted into the canine heart to repair a full-thickness right ventricular defect. As a result, many of the cells spreading from the spheroids expressed cardiac-specific proteins, including sarcomeric α-actinin, cardiotin, and atrial natriuretic peptide, as well as the cell cycle markers cyclin D1 and proliferating cell nuclear antigen. A calcium current similar in amplitude to that of ventricular myocytes was present in 16% of the cells. The cardiogenic cell-seeded scaffolds increased the regional mechanical function in the canine heart compared with the unmanipulated hMSC-seeded scaffolds. In addition, the cells prelabeled with fluorescent markers demonstrated myocyte-specific actinin staining with sarcomere spacing similar to that of normal myocytes. In conclusion, the spheroid-derived cells express cardiac-specific proteins and demonstrate a calcium current similar to adult ventricular myocytes. When these cells are implanted into the canine heart, some of these cells appear striated and mechanical function is improved compared with the unmanipulated hMSCs. Further investigation will be required to determine whether the increased mechanical function is due to a differentiation of the cardiogenic cells to myocytes or to other effects.

The prevention and treatment of life-threatening bradyarrhythmias have been revolutionized in the last half century by electronic pacemakers. Because this represents a palliative therapy, attempts have begun to effect a cure with the novel tools of gene and cell therapy. Over time, the strategies used have coalesced to focus on achieving a stable and autonomically responsive cardiac rhythm in a setting that ultimately would require no implanted hardware. In this report, we review the history of the disease process being treated, approaches now in progress, and the demands that must be met if biological therapies are to be successful.

Cellular cardiomyoplasty has raised hopes of regenerating mechanical function in the heart. Several cell sources have been investigated for their ability to repair the damaged heart, providing reason for optimism. Multiple mechanisms have been proposed for the beneficial effects of the delivered cells; however, true reversal of cardiac damage implies the generation of new contractile myocytes. The assessment of a cell's ability to regenerate contractile cells requires a defined set of criteria that, if met, define success. Here we review data from the four primary players in cellular cardiomyoplasty (skeletal myoblasts, bone marrow cells, embryonic stem cells, and resident cardiac stem cells) and assess their potential to differentiate into contractile myocytes as indicated by their ability to meet such specified milestones. Both animal studies and clinical trials suggest that current experimental approaches to cellular cardiomyoplasty yield short-term improvement, although it may be independent of cell type used. However, the mechanisms underlying this salutary effect, as well as its persistence in the longer term, have remained elusive.

OBJECTIVE—Contraction of cardiac myocytes is initiated by Ca2+ entry through the voltage-dependent l-type Ca2+ channel (LTCC). Previous studies have shown that phosphatidylinositol (PI) 3-kinase signaling modulates LTCC function. Because PI 3-kinases are key mediators of insulin action, we investigated whether LTCC function is affected in diabetic animals due to reduced PI 3-kinase signaling. RESEARCH DESIGN AND METHODS—We used whole-cell patch clamping and biochemical assays to compare cardiac LTCC function and PI 3-kinase signaling in insulin-deficient diabetic mice heterozygous for the Ins2Akita mutation versus nondiabetic littermates. RESULTS—Diabetic mice had a cardiac contractility defect, reduced PI 3-kinase signaling in the heart, and decreased l-type Ca2+ current (ICa,L) density in myocytes compared with control nondiabetic littermates. The lower ICa,L density in myocytes from diabetic mice is due at least in part to reduced cell surface expression of the LTCC. ICa,L density in myocytes from diabetic mice was increased to control levels by insulin treatment or intracellular infusion of PI 3,4,5-trisphosphate [PI(3,4,5)P3]. This stimulatory effect was blocked by taxol, suggesting that PI(3,4,5)P3 stimulates microtubule-dependent trafficking of the LTCC to the cell surface. The voltage dependence of steady-state activation and inactivation of ICa,L was also shifted to more positive potentials in myocytes from diabetic versus nondiabetic animals. PI(3,4,5)P3 infusion eliminated only the difference in voltage dependence of steady-state inactivation of ICa,L. CONCLUSIONS—Decreased PI 3-kinase signaling in myocytes from type 1 diabetic mice leads to reduced Ca2+ entry through the LTCC, which might contribute to the negative effect of diabetes on cardiac contractility.

The voltage-gated potassium channel Kv4.3 was coexpressed with its β-subunit Kv channel-interacting protein 2 and the angiotensin type 1 receptor in HEK-293 cells. Proteomic analysis of proteins coimmunoprecipitated with Kv4.3 revealed that Kv4.3 is associated with Rap guanine nucleotide exchange factors MR-GEF and EPAC-1. Previously, we demonstrated that Kv4.3 interacts with the angiotensin type 1 receptor in HE293 cells and cardiac myocytes. On the basis of this, we investigated the angiotensin type 1 receptor signaling to small G-proteins Ras and Rap-1 in the presence and absence of the Kv4.3–Kv channel-interacting protein 2 macromolecular complex. Ras activation was not significantly affected by coexpression of Kv4.3 and Kv channel-interacting protein 2. Ras exhibited a rapid activation–inactivation pattern with maximum activity at 2.5 min after addition of angiotensin II. In contrast, activation of Rap-1 was affected dramatically by coexpression of Kv4.3 and Kv channel-interacting protein 2 with the angiotensin type 1 receptor. In the absence of Kv4.3 and Kv channel-interacting protein 2, stimulation of the angiotensin type 1 receptor resulted in steady activation of Rap-1 that reached a plateau 25 min after addition of angiotensin II. In the presence of Kv4.3 and Kv channel-interacting protein 2, Rap-1 reaches a maximum activity 2.5 min after addition of angiotensin II and then deactivates rapidly, demonstrating a pattern of activation similar to that of Ras. Our findings show that Kv4.3 regulates angiotensin type 1 receptor signaling to the small G-protein Rap-1.

We investigated effects of the paracrine factors secreted by human mesenchymal stem cells (hMSCs) on endothelial cell migration, extracellular matrix invasion, proliferation, and survival in vitro. Human mesenchymal stem cells were cultured as a monolayer or as three-dimensional aggregates in hanging drops (hMSC spheroids). We performed analysis of paracrine factors in medium conditioned by a monolayer of hMSCs and hMSC spheroids. Concentrations of vascular endothelial growth factor (VEGF), basic fibroblast growth factor, angiogenin, procathepsin B, interleukin (IL)-11, and bone morphogenic protein 2 were increased 5-20 times in medium conditioned by hMSC spheroids, whereas concentrations of IL-6, IL-8, and monocyte hemoattractant protein-1 were not increased. Concentrations of VEGF and angiogenin in medium conditioned by hMSC spheroids showed a weak dependence on the presence of serum, which allows serum-free conditioned medium with elevated concentrations of angiogenic cytokines to be obtained. Medium conditioned by hMSC spheroids was more effective in stimulation of umbilical vein endothelial cell proliferation, migration, and basement membrane invasion than medium conditioned by a monolayer of hMSCs. This medium also promotes endothelial cell survival in vitro. We suggest that culturing of hMSCs as three-dimensional cellular aggregates provides a method to concentrate proangiogenic factors secreted by hMSCs and allows for reduction of serum concentration in conditioned medium. Our data support the hypothesis that hMSCs serve as trophic mediators for endothelial cells. Disclosure of potential conflicts of interest is found at the end of this article.