Knockout Myocytes Research Articles

Regulation of Cardiac L-Type Ca 2+ Current in Na +-Ca 2+ Exchanger Knockout Mice: Functional Coupling of the Ca 2+ Channel and the Na +-Ca 2+ Exchanger

L-type Ca 2+ current ( I Ca) is reduced in myocytes from cardiac-specific Na +-Ca 2+ exchanger (NCX) knockout (KO) mice. This is an important adaptation to prevent Ca 2+ overload in the absence of NCX. However, Ca 2+ channel expression is unchanged, suggesting that regulatory processes reduce I Ca. We tested the hypothesis that an elevation in local Ca 2+ reduces I Ca in KO myocytes. In patch-clamped myocytes from NCX KO mice, peak I Ca was reduced by 50%, and inactivation kinetics were accelerated as compared to wild-type (WT) myocytes. To assess the effects of cytosolic Ca 2+ concentration on I Ca, we used Ba 2+ instead of Ca 2+ as the charge carrier and simultaneously depleted sarcoplasmic reticular Ca 2+ with thapsigargin and ryanodine. Under these conditions, we observed no significant difference in Ba 2+ current between WT and KO myocytes. Also, dialysis with the fast Ca 2+ chelator BAPTA eliminated differences in both I Ca amplitude and decay kinetics between KO and WT myocytes. We conclude that, in NCX KO myocytes, Ca 2+-dependent inactivation of I Ca reduces I Ca amplitude and accelerates current decay kinetics. We hypothesize that the elevated subsarcolemmal Ca 2+ that results from the absence of NCX activity inactivates some L-type Ca 2+ channels. Modulation of subsarcolemmal Ca 2+ by the Na +-Ca 2+ exchanger may be an important regulator of excitation-contraction coupling.

Phospholemman Phosphorylation Mediates the Protein Kinase C–Dependent Effects on Na + /K + Pump Function in Cardiac Myocytes

Because phospholemman (PLM) regulates the Na(+)/K(+) pump (NKA) and is a major cardiac phosphorylation target for both protein kinase A (at Ser68) and protein kinase C (PKC) (at both Ser63 and Ser68), we evaluated whether PLM mediates the PKC-dependent regulation of NKA function and protein kinase A/PKC crosstalk in ventricular myocytes. PKC was activated by PDBu (300 nmol/L), and we measured NKA-mediated [Na(+)](i) decline (fluorescence measurements) and current (I(pump)) (voltage clamp). In wild-type mouse myocytes, PDBu increased PLM phosphorylation at Ser63 and Ser68, I(pump) (both at 10 and 100 mmol/L Na(+) in the pipette solution) and maximal NKA-mediated Na(+) extrusion rate (V(max)) from 7.9+/-1.1 to 12.7+/-1.9 mmol.L(-1) per minute without altering NKA affinity for internal Na(+) (K(0.5)). In PLM knockout mice, PDBu had no effect on either V(max) or K(0.5). After pretreatment with isoproterenol (ISO) (1 mumol/L), PDBu still increased the NKA V(max) and PLM phosphorylation at Ser63 and Ser68. Conversely, after pretreatment with PDBu, ISO further increased the Na(+) affinity of NKA and phosphorylation at Ser68, as it did alone without PDBu. The final NKA activity was independent of the application sequence. The NKA activity in PLM knockout myocytes, after normalizing the protein level, was similar to that after PDBu and ISO treatment. We conclude that (1) PLM mediates the PKC-dependent activation of NKA function in cardiac myocytes, (2) PDBu and ISO effects are additive in the mouse (affecting mainly V(max) and K(0.5), respectively), and (3) PDBu and ISO combine to activate NKA in wild-type to the level found in the PLM knockout mouse.

Mechanism of shortened action potential duration in Na+-Ca2+ exchanger knockout mice

In cardiac-specific Na(+)-Ca(2+) exchanger (NCX) knockout (KO) mice, the ventricular action potential (AP) is shortened. The shortening of the AP, as well as a decrease of the L-type Ca(2+) current (I(Ca)), provides a critical mechanism for the maintenance of Ca(2+) homeostasis and contractility in the absence of NCX (Pott C, Philipson KD, Goldhaber JI. Excitation-contraction coupling in Na(+)-Ca(2+) exchanger knockout mice: reduced transsarcolemmal Ca(2+) flux. Circ Res 97: 1288-1295, 2005). To investigate the mechanism that underlies the accelerated AP repolarization, we recorded the transient outward current (I(to)) in patch-clamped myocytes isolated from wild-type (WT) and NCX KO mice. Peak I(to) was increased by 78% and decay kinetics were slowed in KO vs. WT. Consistent with increased I(to), ECGs from KO mice exhibited shortened QT intervals. Expression of the I(to)-generating K(+) channel subunit Kv4.2 and the K(+) channel interacting protein was increased in KO. We used a computer model of the murine AP (Bondarenko VE, Szigeti GP, Bett GC, Kim SJ, and Rasmusson RL. Computer model of action potential of mouse ventricular myocytes. Am J Physiol Heart Circ Physiol 287: 1378-1403, 2004) to determine the relative contributions of increased I(to), reduced I(Ca), and reduced NCX current (I(NCX)) on the shape and kinetics of the AP. Reduction of I(Ca) and elimination of I(NCX) had relatively small effects on the duration of the AP in the computer model. In contrast, AP repolarization was substantially accelerated when I(to) was increased in the computer model. Thus, the increase in I(to), and not the reduction of I(Ca) or I(NCX), is likely to be the major mechanism of AP shortening in KO myocytes. The upregulation of I(to) may comprise an important regulatory mechanism to limit Ca(2+) influx via a reduction of AP duration, thus preventing Ca(2+) overload in situations of reduced myocyte Ca(2+) extrusion capacity.

Excitation–Contraction Coupling in Na + –Ca 2+ Exchanger Knockout Mice

Cardiac-specific Na+-Ca2+ exchanger (NCX) knockout (KO) mice surprisingly survive into adulthood without compensatory changes in protein expression levels. To determine how cardiac function is maintained in the absence of NCX, we investigated membrane currents, intracellular Ca2+, and action potentials (APs) in whole cell patch-clamped myocytes from wild-type (WT) and NCX knockout mice. There was no difference in resting Ca2+ or sarcoplasmic reticular Ca2+ load between KO and WT. During prolonged caffeine exposure, the decrease of the Ca2+ transient was drastically slowed in KO versus WT myocytes, indicating that no alternative Ca2+-extrusion mechanism is upregulated to compensate for the absence of NCX. Peak L-type Ca2+ current (ICa) was reduced by 62% in KO myocytes compared with WT. Nevertheless, the corresponding Ca2+ transients were similar, implying an increase in the gain of excitation-contraction coupling in KO cells. APs recorded from KO cells repolarized more rapidly than in WT. In WT myocytes, applying a KO AP waveform voltage clamp reduced Ca2+ influx via ICa by 59% compared with WT AP waveform clamps. Again, the corresponding Ca2+ transients remained similar. Our findings indicate that NCX KO myocytes limit Ca2+ influx to &20% of that in WT by reducing ICa and by abbreviating the AP. Contractility is maintained by an increase in the gain of excitation-contraction coupling resulting from both a more rapid repolarization of the AP and an as yet unidentified AP-independent mechanism.

Phosphodiesterase-5 Inhibitor Sildenafil Preconditions Adult Cardiac Myocytes against Necrosis and Apoptosis: ESSENTIAL ROLE OF NITRIC OXIDE SIGNALING

We investigated the effect of sildenafil in protection against necrosis or apoptosis in cardiomyocytes. Adult mouse ventricular myocytes were treated with sildenafil (1 or 10 microM) for 1 h before 40 min of simulated ischemia (SI). Necrosis was determined by trypan blue exclusion and lactate dehydrogenase release following SI alone or plus 1 or 18 h of reoxygenation (RO). Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling assay and mitochondrial membrane potential measured using a fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Sildenafil reduced necrosis as indicated by decrease in trypan blue-positive myocytes and leakage of lactate dehydrogenase compared with untreated cells after either SI or SI-RO. The number of terminal deoxynucleotidyl transferase-mediated nick end labeling-positive myocytes or loss of JC-1 fluorescence following SI and 18 h of RO was attenuated in the sildenafil-treated group with concomitant inhibition of caspase 3 activity. An early increase in Bcl-2 to Bax ratio with sildenafil treatment was also observed in myocytes after SI-RO. The increase of Bcl-2 expression by sildenafil was inhibited by nitric-oxide synthase (NOS) inhibitor, L-nitro-amino-methyl-ester. The drug also enhanced mRNA and protein content of inducible NOS (iNOS) and endothelial NOS (eNOS) in the myocytes. Sildenafil-induced protection against necrosis and apoptosis was absent in the myocytes derived from iNOS knock-out mice and was attenuated in eNOS knock-out myocytes. The up-regulation of Bcl-2 expression by sildenafil was also absent in iNOS-deficient myocytes. Reverse transcription-PCR, Western blots, and immunohistochemical assay confirmed the expression of phosphodiesterase-5 in mouse cardiomyocytes. These data provide strong evidence for a direct protective effect of sildenafil against necrosis and apoptosis through NO signaling pathway. The results may have possible therapeutic potential in preventing myocyte cell death following ischemia/reperfusion.

Binding of myosin binding protein-C to myosin subfragment S2 affects contractility independent of a tether mechanism.

Mutations in the cardiac myosin binding protein-C gene (cMyBP-C) are among the most prevalent causes of inherited hypertrophic cardiomyopathy. Although most cMyBP-C mutations cause reading frameshifts that are predicted to encode truncated peptides, it is not known if or how expression of these peptides causes disease. One possibility is that because the N-terminus contains a unique binding site for the S2 subfragment of myosin, shortened cMyBP-C peptides could directly affect myosin contraction by binding to S2. To test this hypothesis, we compared the effects of a C1C2 protein containing the myosin S2 binding site on contractile properties in permeabilized myocytes from wild-type and cMyBP-C knockout mice. In wild-type myocytes, the C1C2 protein reversibly increased myofilament Ca2+ sensitivity of tension, but had no effect on resting tension. Identical results were observed in cMyBP-C knockout myocytes where C1C2 increased Ca2+ sensitivity of tension with the half-maximal response elicited at approximately 5 micromol/L C1C2. Maximum force was not affected by C1C2. However, phosphorylation of C1C2 by cAMP-dependent protein kinase reduced its ability to increase Ca2+ sensitivity. These results demonstrate that binding of the C1C2 peptide to S2 alone is sufficient to affect myosin contractile function and suggest that regulated binding of cMyBP-C to myosin S2 by phosphorylation directly influences myofilament Ca2+ sensitivity.

Preservation of Base-line Hemodynamic Function and Loss of Inducible Cardioprotection in Adult Mice Lacking Protein Kinase Cϵ

Signaling pathways involving protein kinase C isozymes are modulators of cardiovascular development and response to injury. Protein kinase C epsilon activation in cardiac myocytes reduces necrosis caused by coronary artery disease. However, it is unclear whether protein kinase C epsilon function is required for normal cardiac development or inducible protection against oxidative stress. Protein kinase C delta activation is also observed during cardiac preconditioning. However, its role as a promoter or inhibitor of injury is controversial. We examined hearts from protein kinase C epsilon knock-out mice under physiological conditions and during acute ischemia reperfusion. Null-mutant and wild-type mice displayed equivalent base-line morphology and hemodynamic function. Targeted disruption of the protein kinase C epsilon gene blocked cardioprotection caused by ischemic preconditioning and alpha(1)-adrenergic receptor stimulation. Protein kinase C delta activation increased in protein kinase C epsilon knock-out myocytes without altering resistance to injury. These observations support protein kinase C epsilon activation as an essential component of cardioprotective signaling. Our results favor protein kinase C delta activation as a mediator of normal growth. This study advances the understanding of cellular mechanisms responsible for preservation of myocardial integrity as potential targets for prevention and treatment of ischemic heart disease.

The PDZ-binding motif of the beta2-adrenoceptor is essential for physiologic signaling and trafficking in cardiac myocytes.

beta1- and beta2-adrenergic receptors (AR) regulate cardiac myocyte function through distinct signaling pathways. In addition to regulating cardiac rate and contractility, beta1AR and beta2AR may play different roles in the pathogenesis of heart failure. Studies on neonatal cardiac myocytes from beta1AR and beta2AR knockout mice suggest that subtype-specific signaling is determined by subtype-specific membrane targeting and trafficking. Stimulation of beta2ARs has a biphasic effect on contraction rate, with an initial increase followed by a sustained Gi-dependent decrease. Recent studies show that a PDZ domain-binding motif at the carboxyl terminus of human beta2AR interacts with ezrin-binding protein 50/sodium-hydrogen exchanger regulatory factor, a PDZ-domain-containing protein. The human beta2AR carboxyl terminus also binds to N-ethylmaleimide-sensitive factor, which does not contain a PDZ domain. We found that mutation of the three carboxyl-terminal amino acids in the mouse beta2AR (beta2AR-AAA) disrupts recycling of the receptor after agonist-induced internalization in cardiac myocytes. Nevertheless, stimulation of the beta2AR-AAA produced a greater contraction rate increase than that of the wild-type beta2AR. This enhanced stimulation of contraction rate can be attributed in part to the failure of the beta2AR-AAA to couple to Gi. We also observed that coupling of endogenous, wild-type beta2AR to Gi in beta1AR knockout myocytes is inhibited by treatment with a membrane-permeable peptide representing the beta2AR carboxyl terminus. These studies demonstrate that association of the carboxyl terminus of the beta2AR with ezrin-binding protein 50/sodium-hydrogen exchanger regulatory factor, N-ethylmaleimide-sensitive factor, or some related proteins dictates physiologic signaling specificity and trafficking in cardiac myocytes.

Contribution of the Kir3.1 Subunit to the Muscarinic-gated Atrial Potassium Channel IKACh

The muscarinic-gated atrial potassium (I(KACh)) channel contributes to the heart rate decrease triggered by the parasympathetic nervous system. I(KACh) is a heteromultimeric complex formed by Kir3.1 and Kir3.4 subunits, although Kir3.4 homomultimers have also been proposed to contribute to this conductance. While Kir3.4 homomultimers evince many properties of I(KACh), the contribution of Kir3.1 to I(KACh) is less well understood. Here, we explored the significance of Kir3.1 using knock-out mice. Kir3.1 knock-out mice were viable and appeared normal. The loss of Kir3.1 did not affect the level of atrial Kir3.4 protein but was correlated with a loss of carbachol-induced current in atrial myocytes. Low level channel activity resembling recombinant Kir3.4 homomultimers was observed in 40% of the cell-attached patches from Kir3.1 knock-out myocytes. Channel activity typically ran down quickly, however, and was not recovered in the inside-out configuration despite the addition of GTP and ATP to the bath. Both Kir3.1 knock-out and Kir3.4 knock-out mice exhibited mild resting tachycardias and blunted responses to pharmacological manipulation intended to activate I(KACh). We conclude that Kir3.1 confers properties to I(KACh) that enhance channel activity and that Kir3.4 homomultimers do not contribute significantly to the muscarinic-gated potassium current.

The ryanodine receptor modulates the spontaneous beating rate of cardiomyocytes during development.

In adult myocardium, the heartbeat originates from the sequential activation of ionic currents in pacemaker cells of the sinoatrial node. Ca(2+) release via the ryanodine receptor (RyR) modulates the rate at which these cells beat. In contrast, the mechanisms that regulate heart rate during early cardiac development are poorly understood. Embryonic stem (ES) cells can differentiate into spontaneously contracting myocytes whose beating rate increases with differentiation time. These cells thus offer an opportunity to determine the mechanisms that regulate heart rate during development. Here we show that the increase in heart rate with differentiation is markedly depressed in ES cell-derived cardiomyocytes with a functional knockout (KO) of the cardiac ryanodine receptor (RyR2). KO myocytes show a slowing of the rate of spontaneous diastolic depolarization and an absence of calcium sparks. The depressed rate of pacemaker potential can be mimicked in wild-type myocytes by ryanodine, and rescued in KO myocytes with herpes simplex virus (HSV)-1 amplicons containing full-length RyR2. We conclude that a functional RyR2 is crucial to the progressive increase in heart rate during differentiation of ES cell-derived cardiomyocytes, consistent with a mechanism that couples Ca(2+) release via RyR before an action potential with activation of an inward current that accelerates membrane depolarization.