Jingdong Li

A βIV-spectrin/CaMKII signaling complex is essential for membrane excitability in mice

Ion channel function is fundamental to the existence of life. In metazoans, the coordinate activities of voltage-gated Na(+) channels underlie cellular excitability and control neuronal communication, cardiac excitation-contraction coupling, and skeletal muscle function. However, despite decades of research and linkage of Na(+) channel dysfunction with arrhythmia, epilepsy, and myotonia, little progress has been made toward understanding the fundamental processes that regulate this family of proteins. Here, we have identified β(IV)-spectrin as a multifunctional regulatory platform for Na(+) channels in mice. We found that β(IV)-spectrin targeted critical structural and regulatory proteins to excitable membranes in the heart and brain. Animal models harboring mutant β(IV)-spectrin alleles displayed aberrant cellular excitability and whole animal physiology. Moreover, we identified a regulatory mechanism for Na(+) channels, via direct phosphorylation by β(IV)-spectrin-targeted calcium/calmodulin-dependent kinase II (CaMKII). Collectively, our data define an unexpected but indispensable molecular platform that determines membrane excitability in the mouse heart and brain.

Death, Cardiac Dysfunction, and Arrhythmias Are Increased by Calmodulin Kinase II in Calcineurin Cardiomyopathy

Background— Activation of cellular Ca2+ signaling molecules appears to be a fundamental step in the progression of cardiomyopathy and arrhythmias. Myocardial overexpression of the constitutively active Ca2+-dependent phosphatase calcineurin (CAN) causes severe cardiomyopathy marked by left ventricular (LV) dysfunction, arrhythmias, and increased mortality rate, but CAN antagonist drugs primarily reduce hypertrophy without improving LV function or risk of death. Methods and Results— We found that activity and expression of a second Ca2+-activated signaling molecule, calmodulin kinase II (CaMKII), were increased in hearts from CAN transgenic mice and that CaMKII-inhibitory drugs improved LV function and suppressed arrhythmias. We devised a genetic approach to “clamp” CaMKII activity in CAN mice to control levels by interbreeding CAN transgenic mice with mice expressing a specific CaMKII inhibitor in cardiomyocytes. We developed transgenic control mice by interbreeding CAN transgenic mice with mice expressing an inactive version of the CaMKII-inhibitory peptide. CAN mice with CaMKII inhibition had reduced risk of death and increased LV and ventricular myocyte function and were less susceptible to arrhythmias. CaMKII inhibition did not reduce transgenic overexpression of CAN or expression of endogenous CaMKII protein or significantly reduce most measures of cardiac hypertrophy. Conclusions— CaMKII is a downstream signal in CAN cardiomyopathy, and increased CaMKII activity contributes to cardiac dysfunction, arrhythmia susceptibility, and longevity during CAN overexpression.

RGS6, a Modulator of Parasympathetic Activation in Heart

Rationale: Parasympathetic regulation of heart rate is mediated by acetylcholine binding to G protein–coupled muscarinic M2 receptors, which activate heterotrimeric Gi/o proteins to promote G protein–coupled inwardly rectifying K+ (GIRK) channel activation. Regulator of G protein signaling (RGS) proteins, which function to inactivate G proteins, are indispensable for normal parasympathetic control of the heart. However, it is unclear which of the more than 20 known RGS proteins function to negatively regulate and thereby ensure normal parasympathetic control of the heart. Objective: To examine the specific contribution of RGS6 as an essential regulator of parasympathetic signaling in heart. Methods and Results: We developed RGS6 knockout mice to determine the functional impact of loss of RGS6 on parasympathetic regulation of cardiac automaticity. RGS6 exhibited a uniquely robust expression in the heart, particularly in sinoatrial and atrioventricular nodal regions. Loss of RGS6 provoked dramatically exaggerated bradycardia in response to carbachol in mice and isolated perfused hearts and significantly enhanced the effect of carbachol on inhibition of spontaneous action potential firing in sinoatrial node cells. Consistent with a role of RGS6 in G protein inactivation, RGS6-deficient atrial myocytes exhibited a significant reduction in the time course of acetylcholine-activated potassium current (IKACh) activation and deactivation, as well as the extent of IKACh desensitization. Conclusions: RGS6 is a previously unrecognized, but essential, regulator of parasympathetic activation in heart, functioning to prevent parasympathetic override and severe bradycardia. These effects likely result from actions of RGS6 as a negative regulator of G protein activation of GIRK channels.

Calmodulin Kinase II Inhibition Shortens Action Potential Duration by Upregulation of K+ Currents

The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by elevated intracellular Ca2+ (Ca2+i), and mice with chronic myocardial CaMKII inhibition (Inh) resulting from transgenic expression of a CaMKII inhibitory peptide (AC3-I) unexpectedly showed action potential duration (APD) shortening. Inh mice exhibit increased L-type Ca2+ current (ICa), because of upregulation of protein kinase A (PKA) activity, and decreased CaMKII-dependent phosphorylation of phospholamban (PLN). We hypothesized that CaMKII is a molecular signal linking Ca2+i to repolarization. Whole cell voltage-clamp recordings revealed that the fast transient outward current (Ito,f) and the inward rectifier current (IK1) were selectively upregulated in Inh, compared with wild-type (WT) and transgenic control, mice. Breeding Inh mice with mice lacking PLN returned Ito,f and IK1 to control levels and equalized the APD and QT intervals in Inh mice to control and WT levels. Dialysis of AC3-I into WT cells did not result in increased Ito,f or IK1, suggesting that enhanced cardiac repolarization in Inh mice is an adaptive response to chronic CaMKII inhibition rather than an acute effect of reduced CaMKII activity. Increasing PKA activity, by cell dialysis with cAMP, or inhibition of PKA did not affect IK1 in WT cells. Dialysis of WT cells with cAMP also reduced Ito,f, suggesting that PKA upregulation does not increase repolarizing K+ currents in Inh mice. These findings provide novel in vivo and cellular evidence that CaMKII links Ca2+i to cardiac repolarization and suggest that PLN may be a critical CaMKII target for feedback regulation of APD in ventricular myocytes.

Ankyrin-B Regulates Kir6.2 Membrane Expression and Function in Heart

Ankyrin polypeptides are critical for normal membrane protein expression in diverse cell types, including neurons, myocytes, epithelia, and erythrocytes. Ankyrin dysfunction results in defects in membrane expression of ankyrin-binding partners (including ion channels, transporters, and cell adhesion molecules), resulting in aberrant cellular function and disease. Here, we identify a new role for ankyrin-B in cardiac cell biology. We demonstrate that cardiac sarcolemmal KATP channels directly associate with ankyrin-B in heart via the KATP channel α-subunit Kir6.2. We demonstrate that primary myocytes lacking ankyrin-B display defects in Kir6.2 protein expression, membrane expression, and function. Moreover, we demonstrate a secondary role for ankyrin-B in regulating KATP channel gating. Finally, we demonstrate that ankyrin-B forms a membrane complex with KATP channels and the cardiac Na/K-ATPase, a second key membrane transporter involved in the cardiac ischemia response. Collectively, our new findings define a new role for cardiac ankyrin polypeptides in regulation of ion channel membrane expression in heart.

Calmodulin kinase ii inhibition enhances ischemic preconditioning by augmenting ATP-sensitive K+ current

Mice with genetic inhibition (AC3-I) of the multifunctional Ca2+/calmodulin dependent protein kinase II (CaMKII) have improved cardiomyocyte survival after ischemia. Some K+ currents are up-regulated in AC3-I hearts, but it is unknown if CaMKII inhibition increases the ATP sensitive K+ current (IKATP) that underlies ischemic preconditioning (IP) and confers resistance to ischemia. We hypothesized increased IKATP was part of the mechanism for improved ventricular myocyte survival during ischemia in AC3-I mice. AC3-I hearts were protected against global ischemia due to enhanced IP compared to wild type (WT) and transgenic control (AC3-C) hearts. IKATP was significantly increased, while the negative regulatory dose-dependence of ATP was unchanged in AC3-I compared to WT and AC3-C ventricular myocytes, suggesting that CaMKII inhibition increased the number of functional IKATP channels available for IP. We measured increased sarcolemmal Kir6.2, a pore-forming IKATP subunit, but not a change in total Kir6.2 in cell lysates or single channel IKATP opening probability from AC3-I compared to WT and AC3-C ventricles, showing CaMKII inhibition increased sarcolemmal IKATP channel expression. There were no differences in mRNA for genes encoding IKATP channel subunits in AC3-I, WT and AC3-C ventricles. The IKATP opener pinacidil (100 M) reduced MI area in WT to match AC3-I hearts, while the IKATP antagonist HMR1098 (30 M) increased MI area to an equivalent level in all groups, indicating that increased IKATP and augmented IP are important for reduced ischemic cell death in AC3-I hearts. Our study results show CaMKII inhibition enhances beneficial effects of IP by increasing IKATP.

Ankyrin-B Protein in Heart Failure IDENTIFICATION OF A NEW COMPONENT OF METAZOAN CARDIOPROTECTION

Background: Ankyrin-B is linked with congenital cardiovascular disease; however, its relevance/regulation in heart failure is unknown. Results: Ankyrin-B is altered in heart failure, regulated by calcium-dependent pathways, and serves critical roles in cardioprotection in response to myocardial injury. Conclusion: Ankyrin-B plays a key role in the cardiac response to injury. Significance: Results define a pathway underlying cardiovascular disease. Ankyrins (ankyrin-R, -B, and -G) are adapter proteins linked with defects in metazoan physiology. Ankyrin-B (encoded by ANK2) loss-of-function mutations are directly associated with human cardiovascular phenotypes including sinus node disease, atrial fibrillation, ventricular tachycardia, and sudden cardiac death. Despite the link between ankyrin-B dysfunction and monogenic disease, there are no data linking ankyrin-B regulation with common forms of human heart failure. Here, we report that ankyrin-B levels are altered in both ischemic and non-ischemic human heart failure. Mechanistically, we demonstrate that cardiac ankyrin-B levels are tightly regulated downstream of reactive oxygen species, intracellular calcium, and the calcium-dependent protease calpain, all hallmarks of human myocardial injury and heart failure. Surprisingly, βII-spectrin, previously thought to mediate ankyrin-dependent modulation in the nervous system and heart, is not coordinately regulated with ankyrin-B or its downstream partners. Finally, our data implicate ankyrin-B expression as required for vertebrate myocardial protection as hearts deficient in ankyrin-B show increased cardiac damage and impaired function relative to wild-type mouse hearts following ischemia reperfusion. In summary, our findings provide the data of ankyrin-B regulation in human heart failure, provide insight into candidate pathways for ankyrin-B regulation in acquired human cardiovascular disease, and surprisingly, implicate ankyrin-B as a molecular component for cardioprotection following ischemia.

Differential effects of phospholamban and Ca2+/calmodulin-dependent kinase II on [Ca2+]i transients in cardiac myocytes at physiological stimulation frequencies

In cardiac myocytes, the activity of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is hypothesized to regulate Ca(2+) release from and Ca(2+) uptake into the sarcoplasmic reticulum via the phosphorylation of the ryanodine receptor 2 and phospholamban (PLN), respectively. We tested the role of CaMKII and PLN on the frequency adaptation of cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients in nearly 500 isolated cardiac myocytes from transgenic mice chronically expressing a specific CaMKII inhibitor, interbred into wild-type or PLN null backgrounds under physiologically relevant pacing conditions (frequencies from 0.2 to 10 Hz and at 37 degrees C). When compared with that of mice lacking PLN only, the combined chronic CaMKII inhibition and PLN ablation decreased the maximum Ca(2+) release rate by more than 50% at 10 Hz. Although PLN ablation increased the rate of Ca(2+) uptake at all frequencies, its combination with CaMKII inhibition did not prevent a frequency-dependent reduction of the amplitude and the duration of the [Ca(2+)](i) transient. High stimulation frequencies in the physiological range diminished the effects of PLN ablation on the decay time constant and on the maximum decay rate of the [Ca(2+)](i) transient, indicating that the PLN-mediated feedback on [Ca(2+)](i) removal is limited by high stimulation frequencies. Taken together, our results suggest that in isolated mouse ventricular cardiac myocytes, the combined chronic CaMKII inhibition and PLN ablation slowed Ca(2+) release at physiological frequencies: the frequency-dependent decay of the amplitude and shortening of the [Ca(2+)](i) transient occurs independent of chronic CaMKII inhibition and PLN ablation, and the PLN-mediated regulation of Ca(2+) uptake is diminished at higher stimulation frequencies within the physiological range.