The association of QTc prolongation with cardiovascular events in cancer patients taking tyrosine kinase inhibitors (TKIs).

OBJECTIVE: To investigate the association between stages of QTc prolongation and the risk of cardiac events among patients on TKIs. METHODS: This was a retrospective cohort study performed at an academic tertiary care center of cancer patients who were taking TKIs or not taking TKIs. Patients with two recorded ECGs between January 1, 2009, and December 31, 2019, were selected from an electronic database. The QTc duration > 450ms was determined as prolonged. The association between QTc prolongation progression and events of cardiovascular disease were compared. RESULTS: This study included a total of 451 patients with 41.2% of patients taking TKIs. During a median follow up period of 3.1 years, 49.5% subjects developed CVD and 5.4% subjects suffered cardiac death in patient using TKIs (n = 186); the corresponding rates are 64.2% and 1.2% for patients not on TKIs (n = 265), respectively. Among patient on TKIs, 4.8% of subjects developed stroke, 20.4% of subjects suffered from heart failure (HF) and 24.2% of subjects had myocardial infarction (MI); corresponding incidence are 6.8%, 26.8% and 30.6% in non-TKIs. When patients were regrouped to TKIs versus non-TKIs with and without diabetes, there was no significant difference in the incidence of cardiac events among all groups. Adjusted Cox proportional hazards models were applied to estimate hazard ratios (HRs) with 95% confidence intervals (CIs). There is a significant increased risk of HF events (HR, 95% CI: 2.12, 1.36-3.32) and MI events (HR, 95% CI: 1.78, 1.16-2.73) during the 1st visit. There are also trends for an increased incidence of cardiac adverse events associated with QTc prolongation among patient with QTc > 450ms, however the difference is not statistically significant. Increased cardiac adverse events in patients with QTc prolongation were reproduced during the 2nd visit and the incidence of heart failure was significantly associated with QTc prolongation(HR, 95% CI: 2.94, 1.73-5.0). CONCLUSION: There is a significant increased QTc prolongation in patients taking TKIs. QTc prolongation caused by TKIs is associated with an increased risk of cardiac events.

The Risk of QTc Prolongation in Non-Diabetic and Diabetic Patients Taking Tyrosine Kinase Inhibitors (TKIs)- A Patient Safety Project at a Private Oncology Practice.

Objective: To assess the prevalence of QTc prolongation in both non-diabetic and diabetic patients on TKIs. Some TKIs have been reported to cause QTc prolongation, which is prevalent in diabetes. However, there is no Risk Evaluation and Mitigation Strategy using series ECG to monitor those patients. Methods: Patients taking TKIs, with two ECGs recorded between 1 January 2010 and 31 December 2017 were selected from the electronic database. The QTc duration >450 ms was determined as prolonged. Percentage of QTc prolongation on participants were compared using Chi-Square test. Results: This study included 313 patients (age 66.1 ± 0.8 years and 57.5% are female) taking TKIs. In non-Diabetic patients, the prevalence of QTc prolongation is 19.1% (n = 253) before and 34.8% (n = 253) after treatment with TKIs (p < 0.001), respectively. In diabetic patients, the prevalence of QTc prolongation is 21.7% (n = 60) before and 40% (n = 60) after treatment with TKIs (p = 0.03), respectively. In addition, we examined the effect of modifying risk factors for cardiovascular disease (CVD) on the prevalence of QTc prolongation caused by TKIs. In non-diabetic patients, the prevalence of QTc prolongation is 33.3% (n = 57) before and 34.2% (n = 196) after risk factors modification (p = 0.91), respectively. In diabetic patients, the prevalence of QTc prolongation is 50% (n = 24) before and 33.3% (n = 36) after risk factors modification (p = 0.20), respectively. Conclusion: Use of TKIs is associated with a significantly increased risk of QTc prolongation for patients, particularly when patients are diabetic. Modification of risk factors for CVD does not significantly affect the prevalence of QTc prolongation caused by TKIs.

Modulating the voltage sensor of a cardiac potassium channel shows antiarrhythmic effects.

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 (KCNQ1+KCNE1), 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 the 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.

Regulation of HCN2 Current by PI3K/Akt Signaling.

It has long been known that heart rate is regulated by the autonomic nervous system. Recently, we demonstrated that the pacemaker current, I f , is regulated by phosphoinositide 3-kinase (PI3K) signaling independently of the autonomic nervous system. Inhibition of PI3K in sinus node (SN) myocytes shifts the activation of I f by almost 16 mV in the negative direction. I f in the SN is predominantly mediated by two members of the HCN gene family, HCN4 and HCN1. Purkinje fibers also possess I f and are an important secondary pacemaker in the heart. In contrast to the SN, they express HCN2 and HCN4, while ventricular myocytes, which do not normally pace, express HCN2 alone. In the current work, we investigated PI3K regulation of HCN2 expressed in HEK293 cells. Treatment with the PI3K inhibitor PI-103 caused a negative shift in the activation voltage and a dramatic reduction in the magnitude of the HCN2 current. Similar changes were also seen in cells treated with an inhibitor of the protein kinase Akt, a downstream effector of PI3K. The effects of PI-103 were reversed by perfusion of cells with phosphatidylinositol 3,4,5-trisphosphate (the second messenger produced by PI3K) or active Akt protein. We identified serine 861 in mouse HCN2 as a putative Akt phosphorylation site. Mutation of S861 to alanine mimicked the effects of Akt inhibition on voltage dependence and current magnitude. In addition, the Akt inhibitor had no effect on the mutant channel. These results suggest that Akt phosphorylation of mHCN2 S861 accounts for virtually all of the observed actions of PI3K signaling on the HCN2 current. Unexpectedly, Akt inhibition had no effect on I f in SN myocytes. This result raises the possibility that diverse PI3K signaling pathways differentially regulate HCN-induced currents in different tissues, depending on the isoforms expressed.

Regulation of heart rate and the pacemaker current by phosphoinositide 3-kinase signaling.

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, I F, while addition of its second messenger, phosphatidylinositol 3,4,5-trisphosphate, induces a positive shift. These shifts in the activation of I F 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.

Prevalence of QT prolongation and associated LVEF changes in diabetic patients over a four-year retrospective time period.

Aim: To evaluate the prevalence and longitudinal changes of prolonged QTc in DM patients admitted to our community hospital, and to determine, if any, its correlation with changes of left ventricular ejection fraction (LVEF). Methods: A retrospective chart review of patients with Type 1 (T1DM) and Type 2 (T2DM) with at least two admissions during a four-year period was performed to identify QTc interval, and LVEF, as measured on transthoracic echocardiogram. Changes in QTc and LVEF between patient hospital admissions were compared. Results: A prolonged QTc interval was found in 66.7% (n = 24) of type 1 and 51.3% (n = 154) type 2 diabetic patients. The QTc interval is progressively increased in both type 1 and type 2 diabetes during follow-up, although it did not reach statistical significance. A total of 62% patients (23 out 37 patients) had a reduction of LVEF during follow-up. Conclusion and Discussion: High prevalence of QTc prolongation was confirmed in hospitalized patients with in both T1DM and T2DM. Significant reduction of LVEF correlated with QTc prolongation over a mean of 17.3 months in T2DM patients, and may have implications for interventions. Abbreviations CHF: Congestive heart failure LVEF: Left ventricular ejection fraction.

Development of a universal RNA beacon for exogenous gene detection.

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.

Bitter tastants induce relaxation of rat thoracic aorta precontracted with high K(+).

It has been reported that bitter tastants decrease blood pressure and relax precontracted vascular smooth muscle. However, the underlying mechanisms remain unclear. The aim of the present study was to determine the mechanism underlying the vasorelaxant effect of the bitter tastants. Thoracic aortic rings were isolated from Wistar rats and contractions were measured using an isometric myograph. Intracellular Ca(2+) ([Ca(2+)]i) in single rat thoracic aortic smooth muscle cells was recorded by calcium imaging. Calcium currents in single cells were recorded using patch-clamp techniques. High K(+) (140 mmol/L) induced contractions in rat thoracic aortic rings that were inhibited by 3 mmol/L chloroquine, 3 mmol/L denatonium and 10 µmol/L nifedipine. In single rat thoracic aortic smooth muscle cells, high K(+) increased [Ca(2+)]i and this effect was also blocked by 3 mmol/L chloroquine and 10 µmol/L nifedipine. Under Ca(2+) -free conditions, high K(+) failed to induce contractions in rat thoracic aortic rings. On its own, chloroquine had no effect on the muscle tension of rat aortic rings and [Ca(2+) ]i. The vasorelaxant effects of chloroquine on precontracted rat thoracic aortic rings were not altered by either 1 µg/mL pertussis toxin (PTX), an inhibitor of Gαo/i-protein, or 1 mmol/L gallein, an inhibitor of Gßγ-protein. The results of patch-clamp analysis in single cells indicate that 1 mmol/L chloroquine blocks voltage-dependent L-type Ca(2+) channel (VDLCC) currents from both extracellular and intracellular sides. Together, the results indicate that chloroquine can block VDLCC, independent of PTX- and gallein-sensitive G-proteins, resulting in relaxation of high K(+)-precontracted thoracic aortic smooth muscle.

Intracellular ATP binding is required to activate the slowly activating K+ channel I(Ks).

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.

Computational optogenetics: empirically-derived voltage- and light-sensitive channelrhodopsin-2 model.

Channelrhodospin-2 (ChR2), a light-sensitive ion channel, and its variants have emerged as new excitatory optogenetic tools not only in neuroscience, but also in other areas, including cardiac electrophysiology. An accurate quantitative model of ChR2 is necessary for in silico prediction of the response to optical stimulation in realistic tissue/organ settings. Such a model can guide the rational design of new ion channel functionality tailored to different cell types/tissues. Focusing on one of the most widely used ChR2 mutants (H134R) with enhanced current, we collected a comprehensive experimental data set of the response of this ion channel to different irradiances and voltages, and used these data to develop a model of ChR2 with empirically-derived voltage- and irradiance- dependence, where parameters were fine-tuned via simulated annealing optimization. This ChR2 model offers: 1) accurate inward rectification in the current-voltage response across irradiances; 2) empirically-derived voltage- and light-dependent kinetics (activation, deactivation and recovery from inactivation); and 3) accurate amplitude and morphology of the response across voltage and irradiance settings. Temperature-scaling factors (Q10) were derived and model kinetics was adjusted to physiological temperatures. Using optical action potential clamp, we experimentally validated model-predicted ChR2 behavior in guinea pig ventricular myocytes. The model was then incorporated in a variety of cardiac myocytes, including human ventricular, atrial and Purkinje cell models. We demonstrate the ability of ChR2 to trigger action potentials in human cardiomyocytes at relatively low light levels, as well as the differential response of these cells to light, with the Purkinje cells being most easily excitable and ventricular cells requiring the highest irradiance at all pulse durations. This new experimentally-validated ChR2 model will facilitate virtual experimentation in neural and cardiac optogenetics at the cell and organ level and provide guidance for the development of in vivo tools.

Increased persistent sodium current due to decreased PI3K signaling contributes to QT prolongation in the diabetic heart.

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.

Microtubules and angiotensin II receptors contribute to modulation of repolarization induced by ventricular pacing.

BACKGROUND: Left ventricular pacing (LVP) in canine heart alters ventricular activation, leading to reduced transient outward potassium current (I(to)), loss of the epicardial action potential notch, and T-wave vector displacement. These repolarization changes, referred to as cardiac memory, are initiated by locally increased angiotensin II (AngII) levels. In HEK293 cells in which Kv4.3 and KChIP2, the channel subunits contributing to I(to), are overexpressed with the AngII receptor 1 (AT1R), AngII induces a decrease in I(to) as the result of internalization of a Kv4.3/KChIP2/AT1R macromolecular complex. OBJECTIVE: To test the hypothesis that in canine heart in situ, 2h LVP-induced decreases in membrane KChIP2, AT1R, and I(to) are prevented by blocking subunit trafficking. METHODS: We used standard electrophysiological, biophysical, and biochemical methods to study 4 groups of dogs: (1) Sham, (2) 2h LVP, (3) LVP + colchicine (microtubule-disrupting agent), and (4) LVP + losartan (AT1R blocker). RESULTS: The T-wave vector displacement was significantly greater in LVP than in Sham and was inhibited by colchicine or losartan. Epicardial biopsies showed significant decreases in KChIP2 and AT1R proteins in the membrane fraction after LVP but not after sham treatment, and these decreases were prevented by colchicine or losartan. Colchicine but not losartan significantly reduced microtubular polymerization. In isolated ventricular myocytes, AngII-induced I(to) reduction and loss of action potential notch were blocked by colchicine. CONCLUSIONS: LVP-induced reduction of KChIP2 in plasma light membranes depends on an AngII-mediated pathway and intact microtubular status. Loss of I(to) and the action potential notch appear to derive from AngII-initiated trafficking of channel subunits.

Improving cardiac conduction with a skeletal muscle sodium channel by gene and cell therapy.

The voltage-gated Na+ channel is a critical determinant of the action potential (AP) upstroke. Increasing Na+ conductance may speed AP propagation. In this study, we propose use of the skeletal muscle Na+ channel SkM1 as a more favorable gene than the cardiac isoform SCN5A to enhance conduction velocity in depolarized cardiac tissue. We used cells that electrically coupled with cardiac myocytes as a delivery platform to introduce the Na+ channels. Human embryonic kidney 293 cells were stably transfected with SkM1 or SCN5A. SkM1 had a more depolarized (18 mV shift) inactivation curve than SCN5A. We also found that SkM1 recovered faster from inactivation than SCN5A. When coupled with SkM1 expressing cells, cultured myocytes showed an increase in the dV/dtmax of the AP. Expression of SCN5A had no such effect. In an in vitro cardiac syncytium, coculture of neonatal cardiac myocytes with SkM1 expressing but not SCN5A expressing cells significantly increased the conduction velocity under both normal and depolarized conditions. In an in vitro reentry model induced by high-frequency stimulation, expression of SkM1 also enhanced angular velocity of the induced reentry. These results suggest that cells carrying a Na+ channel with a more depolarized inactivation curve can improve cardiac excitability and conduction in depolarized tissues.

Suppression of phosphoinositide 3-kinase signaling and alteration of multiple ion currents in drug-induced long QT syndrome.

Many drugs, including some commonly used medications, can cause abnormal heart rhythms and sudden death, as manifest by a prolonged QT interval in the electrocardiogram. Cardiac arrhythmias caused by drug-induced long QT syndrome are thought to result mainly from reductions in the delayed rectifier potassium ion (K(+)) current I(Kr). Here, we report a mechanism for drug-induced QT prolongation that involves changes in multiple ion currents caused by a decrease in phosphoinositide 3-kinase (PI3K) signaling. Treatment of canine cardiac myocytes with inhibitors of tyrosine kinases or PI3Ks caused an increase in action potential duration that was reversed by intracellular infusion of phosphatidylinositol 3,4,5-trisphosphate. The inhibitors decreased the delayed rectifier K(+) currents I(Kr) and I(Ks), the L-type calcium ion (Ca(2+)) current I(Ca,L), and the peak sodium ion (Na(+)) current I(Na) and increased the persistent Na(+) current I(NaP). Computer modeling of the canine ventricular action potential showed that the drug-induced change in any one current accounted for less than 50% of the increase in action potential duration. Mouse hearts lacking the PI3K p110α catalytic subunit exhibited a prolonged action potential and QT interval that were at least partly a result of an increase in I(NaP). These results indicate that down-regulation of PI3K signaling directly or indirectly via tyrosine kinase inhibition prolongs the QT interval by affecting multiple ion channels. This mechanism may explain why some tyrosine kinase inhibitors in clinical use are associated with increased risk of life-threatening arrhythmias.

Restoration of defective L-type Ca2+ current in cardiac myocytes of type 2 diabetic db/db mice by Akt and PKC-ι.

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.

Loss of cardiac phosphoinositide 3-kinase p110 alpha results in contractile dysfunction.

BACKGROUND: Phosphoinositide 3-kinase (PI3K) p110alpha 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 p110alpha also controls cardiac contractility by regulating the LTCC. METHODS AND RESULTS: Genetic ablation of p110alpha (also known as Pik3ca), but not p110beta (also known as Pik3cb), in cardiac myocytes of adult mice reduced I(Ca,L) and blocked insulin signaling in the heart. p110alpha-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 p110alpha decreased I(Ca,L) and contractility in canine myocytes. Inhibition of p110beta did not reduce I(Ca,L). CONCLUSIONS: PI3K p110alpha but not p110beta regulates the LTCC in cardiac myocytes. Decreased signaling to p110alpha reduces the number of LTCCs on the cell surface and thus attenuates I(Ca,L) and contractility.

Enhanced recovery of mechanical function in the canine heart by seeding an extracellular matrix patch with mesenchymal stem cells committed to a cardiac lineage.

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 alpha-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.

Replacing damaged myocardium.

Heart failure survival after diagnosis has barely changed for more than half a century. Recently, investigation has focused on differentiation of stem cells in vitro and their delivery for use in vivo as replacement cardiac contractile elements. Here we report preliminary results using mesenchymal stem cells partially differentiated to a cardiac lineage in vitro. When delivered to the canine heart on an extracellular matrix patch to replace a full-thickness ventricular defect in vivo, they improve regional mechanical function. The delivered cells were also tracked, and some became myocytes with mature sarcomeres.

Two-pore K+ channels, NO and metabolic inhibition.

Ischemic preconditioning is a potent endogenous mechanism protecting many organs from the devastating effects of prolonged ischemia. In the heart, NO is one mediator of this myoprotective response thought to involve activation of the K(ATP) channel. Ischemic preconditioning is known to be induced by metabolic inhibition using sodium cyanide (NaCN) in single cardiomyocytes. In the present study, we show for the first time that the end effector channel activated by NaCN has been incorrectly identified. The channel activated is not K(ATP) but instead belongs to the relatively new family of two-pore domain potassium channels (K2P). Further when activated by metabolic ischemia, the amplitude of K2P current is directly modulated by activators and inhibitors of the NO pathway.

Decreased L-type Ca2+ current in cardiac myocytes of type 1 diabetic Akita mice due to reduced phosphatidylinositol 3-kinase signaling.

OBJECTIVE: Contraction of cardiac myocytes is initiated by Ca(2+) entry through the voltage-dependent L-type Ca(2+) 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 Ins2(Akita) mutation versus nondiabetic littermates. RESULTS: Diabetic mice had a cardiac contractility defect, reduced PI 3-kinase signaling in the heart, and decreased L-type Ca(2+) current (I(Ca,L)) density in myocytes compared with control nondiabetic littermates. The lower I(Ca,L) density in myocytes from diabetic mice is due at least in part to reduced cell surface expression of the LTCC. I(Ca,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)P(3)]. This stimulatory effect was blocked by taxol, suggesting that PI(3,4,5)P(3) stimulates microtubule-dependent trafficking of the LTCC to the cell surface. The voltage dependence of steady-state activation and inactivation of I(Ca,L) was also shifted to more positive potentials in myocytes from diabetic versus nondiabetic animals. PI(3,4,5)P(3) infusion eliminated only the difference in voltage dependence of steady-state inactivation of I(Ca,L). CONCLUSIONS: Decreased PI 3-kinase signaling in myocytes from type 1 diabetic mice leads to reduced Ca(2+) entry through the LTCC, which might contribute to the negative effect of diabetes on cardiac contractility.