Christopher A. Ward

Chronic Phospholamban–Sarcoplasmic Reticulum Calcium ATPase Interaction Is the Critical Calcium Cycling Defect in Dilated Cardiomyopathy

Dilated cardiomyopathy and end-stage heart failure result in multiple defects in cardiac excitation-contraction coupling. Via complementation of a genetically based mouse model of dilated cardiomyopathy, we now provide evidence that progressive chamber dilation and heart failure are dependent on a Ca2+ cycling defect in the cardiac sarcoplasmic reticulum. The ablation of a muscle-specific sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) inhibitor, phospholamban, rescued the spectrum of phenotypes that resemble human heart failure. Inhibition of phospholamban-SERCA2a interaction via in vivo expression of a phospholamban point mutant dominantly activated the contractility of ventricular muscle cells. Thus, interfering with phospholamban-SERCA2a interaction may provide a novel therapeutic approach for preventing the progression of dilated cardiomyopathy.

PKA phosphorylation activates the calcium release channel (ryanodine receptor) in skeletal muscle: defective regulation in heart failure

The type 1 ryanodine receptor (RyR1) on the sarcoplasmic reticulum (SR) is the major calcium (Ca2+) release channel required for skeletal muscle excitation–contraction (EC) coupling. RyR1 function is modulated by proteins that bind to its large cytoplasmic scaffold domain, including the FK506 binding protein (FKBP12) and PKA. PKA is activated during sympathetic nervous system (SNS) stimulation. We show that PKA phosphorylation of RyR1 at Ser2843 activates the channel by releasing FKBP12. When FKB12 is bound to RyR1, it inhibits the channel by stabilizing its closed state. RyR1 in skeletal muscle from animals with heart failure (HF), a chronic hyperadrenergic state, were PKA hyperphosphorylated, depleted of FKBP12, and exhibited increased activity, suggesting that the channels are “leaky.” RyR1 PKA hyperphosphorylation correlated with impaired SR Ca2+ release and early fatigue in HF skeletal muscle. These findings identify a novel mechanism that regulates RyR1 function via PKA phosphorylation in response to SNS stimulation. PKA hyperphosphorylation of RyR1 may contribute to impaired skeletal muscle function in HF, suggesting that a generalized EC coupling myopathy may play a role in HF.

Emigrated Neutrophils Regulate Ventricular Contractility via α4 Integrin

We have previously shown that CD18 and alpha4 integrin were important in the adherence of emigrated neutrophils to cardiac myocytes. Whether either of these molecules is important in myocyte dysfunction is unclear. In this study, we measured contractility as an index of myocyte function. Control contractility was compared with shortening response in myocytes exposed to neutrophils in the presence and absence of anti-CD18 or anti-alpha4 antibodies. Control unloaded cell shortening, expressed as a percentage of resting cell length, measured 10.06+/-1.16% (n=10) at 5 minutes. Circulating neutrophils caused a 35% reduction in cell shortening, an event prevented by anti-CD18, but not by anti-alpha4 antibody. When emigrated neutrophils were added to the myocytes, a profound reduction (50%) in unloaded cell shortening was noted. A significant increase in CD18 and alpha4 integrin was found on emigrated neutrophils. Addition of anti-CD18 antibody did not protect the myocyte from the emigrated neutrophils, whereas the addition of an anti-alpha4 antibody significantly reduced neutrophil-induced cell shortening, despite some neutrophils still adhering to the myocytes. Furthermore, emigrated neutrophils were able to cause myocytes to go into contracture within 5 minutes in the presence of neutrophils with or without anti-CD18 antibody. In addition to the impairment in unloaded cell shortening, at later times (10 minutes), neutrophils also caused a 40% reduction in the rate of contraction and relaxation. The addition of either anti-CD18 or anti-alpha4 antibody protected the myocytes from these changes. The data suggest that immunosuppression of CD18 on emigrated neutrophils was only partially effective in reducing myocyte dysfunction. In contrast, immunosuppression of the alpha4 integrin alone was sufficient to dramatically reduce all parameters of cell dysfunction measured in this study.

Distinct phosphodiesterase-4D variants integrate into protein kinase A-based signaling complexes in cardiac and vascular myocytes.

Numerous cAMP-elevating agents regulate events required for efficient migration of arterial vascular smooth muscle cells (VSMCs). Interestingly, when the impact of cAMP-elevating agents on individual migration-related events is studied, these agents have been shown to have distinct, and sometimes unexpected, effects. For example, although cAMP-elevating agents inhibit overall migration, they promote VSMC adhesion to extracellular matrix proteins and the formation of membrane extensions, which are both events that are essential for and promote migration. Herein, we extend previous observations that identified phosphodiesterase-4D3 (PDE4D3) as an integral component of a PKA/A kinase-anchoring protein (AKAP) complex in cultured/hypertrophied rat cardiac myocytes to the case for nonhypertrophied cardiac myocytes. Moreover, we show that while rat aortic VSMCs also express PDE4D3, this protein is not detected in PKA/AKAP complexes isolated from these cells. In contrast, we show that another PDE4D splice variant expressed in arterial vascular myocytes, namely, PDE4D8, integrates into PKA/AKAP-based signaling complexes in VSMCs. Consistent with the idea that a PDE4D8/PKA/AKAP complex regulates specific VSMC functions, PKA and PDE4D8 were each recruited to leading-edge structures in migrating VSMCs, and inhibition of PDE4D8 recruitment to pseudopodia of migrating cells caused localized changes in actin dynamics. Our data are presented in the context that cardiac myocytes and arterial VSMCs may use distinct PDE4D variants to regulate selected pools of targeted PKA activity and that disruption of this complex may allow selective regulation of cAMP-dependent events between these two cardiovascular cell types.

Block of Na+ channel by bepridil in isolated guinea-pig ventricular myocytes.

The effects of bepridil, a potent antiarrhythmic agent, on the Na+ current (INa) of single guinea-pig ventricular myocytes were studied using the whole-cell patch-clamp technique. Bepridil inhibited INa in a dose-dependent manner without causing any change in the I-V. relationship for INa. Bepridil suppressed INa with Kd values of 342 and 40 microM when cells were clamped to holding potentials of -140 and -90 mV, respectively. 10 microM bepridil shifted the steady-state inactivation curve for INa toward more negative potentials by 7.7 mV (n = 6). Bepridil also produced marked use-dependent block with a rapid onset. Recovery of INa from inactivation was retarded (time constant 290 ms) at a holding potential of -140 mV in the presence of 10 microM bepridil. When the onset of INa block was studied in experiments using a double-pulse protocol, bepridil blocked INa by 11.5% after a 4-ms pre-pulse, but significantly blocked it after pre-pulses longer than 16 ms. These results suggest that: (1) bepridil has a higher affinity for the inactivated state than the resting state of Na+ channel; (2) the drug also produces an open channel block; and (3) the drug shows a lidocaine-like fast kinetic block of Na+ current.

Effects of Phloretin and Phloridzin on Ca2+ Handling, the Action Potential, and Ion Currents in Rat Ventricular Myocytes

The effects of the phytoestrogens phloretin and phloridzin on Ca2+ handling, cell shortening, the action potential, and Ca2+ and K+ currents in freshly isolated cardiac myocytes from rat ventricle were examined. Phloretin increased the amplitude and area and decreased the rate of decline of electrically evoked Ca2+ transients in the myocytes. These effects were accompanied by an increase in the Ca2+ load of the sarcoplasmic reticulum, as determined by the area of caffeine-evoked Ca2+ transients. An increase in the extent of shortening of the myocytes in response to electrically evoked action potentials was also observed in the presence of phloretin. To further examine possible mechanisms contributing to the observed changes in Ca2+ handling and contractility, the effects of phloretin on the cardiac action potential and plasma membrane Ca2+ and K+ currents were examined. Phloretin markedly increased the action potential duration in the myocytes, and it inhibited the Ca2+-independent transient outward K+ current (Ito). The inwardly rectifying K+ current, the sustained outward delayed rectifier K+ current, and L-type Ca2+ currents were not significantly different in the presence and absence of phloretin, nor was there any evidence that the Na+/Ca2+ exchanger was affected. The effects of phloretin on Ca2+ handling in the myocytes are consistent with its effects on Ito. Phloridzin did not significantly alter the amplitude or area of electrically evoked Ca2+ transients in the myocytes, nor did it have detectable effects on the sarcoplasmic reticulum Ca2+ load, cell shortening, or the action potential.