Scott A. Henderson

Functional Adult Myocardium in the Absence of Na+-Ca2+ Exchange. Cardiac-Specific Knockout of NCX1

The excitation–contraction coupling cycle in cardiac muscle is initiated by an influx of Ca2+ through voltage-dependent Ca2+ channels. Ca2+ influx induces a release of Ca2+ from the sarcoplasmic reticulum and myocyte contraction. To maintain Ca2+ homeostasis, Ca2+ entry is balanced by efflux mediated by the sarcolemmal Na+-Ca2+ exchanger. In the absence of Na+-Ca2+ exchange, it would be expected that cardiac myocytes would overload with Ca2+. Using Cre/loxP technology, we generated mice with a cardiac-specific knockout of the Na+-Ca2+ exchanger, NCX1. The exchanger is completely ablated in 80% to 90% of the cardiomyocytes as determined by immunoblot, immunofluorescence, and exchange function. Surprisingly, the NCX1 knockout mice live to adulthood with only modestly reduced cardiac function as assessed by echocardiography. At 7.5 weeks of age, measures of contractility are decreased by 20% to 30%. We detect no adaptation of the myocardium to the absence of the Na+-Ca2+ exchanger as measured by both immunoblots and microarray analysis. Ca2+ transients of isolated myocytes from knockout mice display normal magnitudes and relaxation kinetics and normal responses to isoproterenol. Under voltage clamp conditions, the current through L-type Ca2+ channels is reduced by 50%, although the number of channels is unchanged. An abbreviated action potential may further reduce Ca2+ influx. Rather than upregulate other Ca2+ efflux mechanisms, the myocardium appears to functionally adapt to the absence of the Na+-Ca2+ exchanger by limiting Ca2+ influx. The magnitude of Ca2+ transients appears to be maintained by an increased gain of sarcoplasmic reticular Ca2+ release. The myocardium of the NCX1 knockout mice undergoes a remarkable adaptation to maintain near normal cardiac function.

Knockout mice for pharmacological screening: testing the specificity of Na+-Ca2+ exchange inhibitors.

The role of the Na+-Ca2+ exchanger as a major determinant of cell Ca2+ is well defined in cardiac tissue, and there has been much effort to develop specific inhibitors of the exchanger. We use a novel system to test the specificity of two putative specific inhibitors, KB-R7943 and SEA0400. The drugs are applied to electrically stimulated heart tubes from control mouse embryos or embryos with the Na+-Ca2+ exchanger knocked out. We monitored effects of the drugs on Ca2+ transients. Both drugs depress the Ca2+ transients at low concentrations even in the absence of any Na+-Ca2+ exchanger. KB-R7943 and SEA0400 are not completely specific and should be used with caution as Na+-Ca2+ exchange inhibitors.

Cardiac-Myocyte-Specific Excision of the Vinculin Gene Disrupts Cellular Junctions, Causing Sudden Death or Dilated Cardiomyopathy

ABSTRACT Vinculin is a ubiquitously expressed multiliganded protein that links the actin cytoskeleton to the cell membrane. In myocytes, it is localized in protein complexes which anchor the contractile apparatus to the sarcolemma. Its function in the myocardium remains poorly understood. Therefore, we developed a mouse model with cardiac-myocyte-specific inactivation of the vinculin (Vcl) gene by using Cre-loxP technology. Sudden death was found in 49% of the knockout (cVclKO) mice younger than 3 months of age despite preservation of contractile function. Conscious telemetry documented ventricular tachycardia as the cause of sudden death, while defective myocardial conduction was detected by optical mapping. cVclKO mice that survived through the vulnerable period of sudden death developed dilated cardiomyopathy and died before 6 months of age. Prior to the onset of cardiac dysfunction, ultrastructural analysis of cVclKO heart tissue showed abnormal adherens junctions with dissolution of the intercalated disc structure, expression of the junctional proteins cadherin and β1D integrin were reduced, and the gap junction protein connexin 43 was mislocalized to the lateral myocyte border. This is the first report of tissue-specific inactivation of the Vcl gene and shows that it is required for preservation of normal cell-cell and cell-matrix adhesive structures.

Prostaglandin F2α Stimulates Hypertrophic Growth of Cultured Neonatal Rat Ventricular Myocytes

Prostaglandin F2α (PGF2α) stimulates protein synthesis of skeletal and smooth muscle cells in culture and is elevated in the heart during compensatory growth. We hypothesized that PGF2α stimulates hypertrophic growth of neonatal rat cardiac myocytes. Prostaglandin F2α increased [3H]phenylalanine incorporation by cultured ventricular myocytes in a dose-dependent manner (EC50 = 11 nM), suggesting action through a PGF-specific receptor. Semiquantitative reverse transcriptase polymerase chain reaction revealed that PGF receptor mRNA is expressed in ventricular myocytes > A7R5 vascular smooth muscle cells ≫ cardiac fibroblast-like cells. The protein content of cardiomyocyte cultures was increased by 10 nM PGF2α and 11β-PGF2α but was unchanged by 10 nM PGD2, PGE2, PGF1α, carbaprostacyclin, U-46619, or 12- or 15-hydroxyeicosatrienoic acid. Stimulation of myofibrillar gene expression by PGF2α was demonstrated by Northern and Western blot analysis for myosin light chain-2 (MLC-2) and by transient transfection experiments with MLC-2 luciferase expression plasmids. In addition, myofibrillogenesis was increased by PGF2α as assessed by immunocytochemical staining with MLC-2 antisera. Prostaglandin F2α did not affect myocyte proliferation or [3H]thymidine incorporation, thus myocyte growth occurred by hypertrophy. Proliferative and hypertrophic growth of cardiac fibroblast-like cells were unaffected by PGF2α. We conclude that PGF2α stimulates hypertrophic growth of neonatal rat ventricular myocytes in culture and speculate that PGF2α plays a role in myocardial adaptation to chronic hypertrophic stimuli, recovery from injury, and cardiac ontogeny.

Phorbol esters induce immediate-early genes and activate cardiac gene transcription in neonatal rat myocardial cells

The mechanisms which transduce intracellular signals for the accumulation of myofibrillar protein during the onset of myocardial cell hypertrophy are unknown. Although previous studies in skeletal muscle cells have suggested that the activation of protein kinase C induces de-differentiation, including the selective disassembly of myofibrils and inhibition of myofibrillar protein synthesis, the present study demonstrates that phorbol esters which activate protein kinase C lead to the accumulation of an individual contractile protein, myosin light chain-2 (MLC-2) and produce several features of myocardial cell hypertrophy. Utilizing immunoblotting and indirect immunocytofluorescence with MLC antisera, the present study demonstrates a several-fold increase in the content of MLC-2, and a marked increase in the assembly of MLC into organized contractile units in individual neonatal rat myocardial cells following treatment with phorbol 12-myristate 13-acetate (PMA). The concentration of PMA required to elicit this response and the lack of a response with an inactive phorbol ester is consistent with the activation of a protein kinase C dependent pathway. Furthermore, PMA treatment results in the rapid induction of a program of immediate-early gene expression (including the c-fos and c-jun proto-oncogenes, and an inducible zinc finger containing gene, egr-l), and activates cardiac gene transcription as assessed by nuclear run-on analyses. The results of the present study suggest the possibility that a protein kinase C dependent pathway may be involved in the up-regulation of myofibrillar protein content and the activation of cardiac gene transcription during growth and hypertrophy of neonatal rat myocardium, and that the induction of a program of immediate-early gene expression may be linked to this response.

Impaired Contractile Performance of Cultured Rabbit Ventricular Myocytes After Adenoviral Gene Transfer of Na+-Ca2+ Exchanger

Na+-Ca2+ exchanger (NCX) gene expression is increased in the failing human heart. We investigated the hypothesis that upregulation of NCX can induce depressed contractile performance. Overexpression of NCX was achieved in isolated rabbit ventricular myocytes through adenoviral gene transfer (Ad-NCX). After 48 hours, immunoblots revealed a virus dose-dependent increase in NCX protein. Adenoviral &bgr;-galactosidase transfection served as a control. The fractional shortening (FS) of electrically stimulated myocytes was analyzed. At 60 min−1, FS was depressed by 15.6% in the Ad-NCX group (n=143) versus the control group (n=163, P <0.05). Analysis of the shortening-frequency relationship showed a steady increase in FS in the control myocytes (n=26) from 0.027±0.002 at 30 min−1 to 0.037±0.002 at 120 min−1 (P <0.05 versus 30 min−1) and to 0.040±0.002 at 180 min−1 (P <0.05 versus 30 min−1). Frequency potentiation of shortening was blunted in NCX-transfected myocytes (n=27). The FS was 0.024±0.002 at 30 min−1, 0.029±0.002 at 120 min−1 (P <0.05 versus 30 min−1, P <0.05 versus control), and 0.026±0.002 at 180 min−1 (NS versus 30 min−1, P <0.05 versus control). Caffeine contractures, which indicate sarcoplasmic reticulum Ca2+ load, were significantly reduced at 120 min−1 in NCX-transfected cells. An analysis of postrest behavior showed a decay of FS with longer rest intervals in control cells. Rest decay was significantly higher in the Ad-NCX group; after 120 seconds of rest, FS was 78±4% in control and 65±3% in the Ad-NCX group (P <0.05) relative to steady-state FS before rest (100%). In conclusion, the overexpression of NCX in rabbit cardiomyocytes results in the depression of contractile function. This supports the hypothesis that upregulation of NCX can result in systolic myocardial failure.

Tyrosine Kinase and c-Jun NH2-Terminal Kinase Mediate Hypertrophic Responses to Prostaglandin F2α in Cultured Neonatal Rat Ventricular Myocytes

Myocardial infarction results in focal areas of ischemia, hypoxia, necrosis, and decreased contractile function. To compensate for loss of contractile function, remaining viable myocytes undergo hypertrophic growth. Prostaglandin F2alpha (PGF2alpha), which is released from cells of the myocardium during periods of stress such as hypoxia or ischemia/reperfusion, has recently been shown to stimulate hypertrophic growth in neonatal rat ventricular myocytes. In the present study, we determine which growth-related intracellular pathways are required for PGF2alpha to induce morphological and genetic features characteristic of the hypertrophic phenotype. In cardiomyocytes, PGF2alpha increases the hydrolysis of inositol phosphates and induces the translocation of protein kinase C epsilon to the myocyte membrane, consistent with PGF2alpha receptor coupling to Gq. PGF2alpha also activates the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase pathways. Surprisingly, studies using pharmacological inhibitors and transfection of dominant-interfering proteins demonstrate that PGF2alpha-induced myocyte hypertrophy occurs independent of either PKC, p38, or ERK pathways. Additional studies demonstrate that PGF2alpha stimulates protein tyrosine phosphorylation and activates c-Jun NH2-terminal kinase and suggest that these pathways mediate hypertrophic growth in response to PGF2alpha.

Cardiac excitation-contraction coupling in the absence of Na(+) - Ca2+ exchange.

We investigate cardiac excitation-contraction coupling in the absence of sarcolemmal Na(+) - Ca(2+) exchange using NCX1 knock out mice. Knock out of NCX1 is embryonic lethal, and we measure Ca(2+) transients and contractions in heart tubes from embryos at day 9.5 post coitum. Immunoblot and electron microscopy both indicate that sarcoplasmic reticular membranes are diminished in the knock out (NCX(-/-)) heart tubes. Both Ni(2+) and nifedipine block excitation-contraction coupling in NCX-containing (NCX+) and NCX(-/-) heart tubes indicating an essential role for the L-type Ca(2+) current. Under basal conditions (1Hz stimulation), the NCX(-/-) heart tubes have normal Ca(2+) transients but are unable to maintain homeostasis when Ca(2+) fluxes are increased by various interventions (increased stimulation frequency, caffeine, isoproterenol). In each case, the NCX(-/-) heart tubes respond to the intervention in a more deleterious manner (increased diastolic Ca(2+), decreased Ca(2+) transient) than the NCX+ heart tubes. Expression of the sarcolemmal Ca(2+) pump was not upregulated. The sarcolemmal Ca(2+) pump, however, was able to compensate surprisingly well for the absence of Na(+) - Ca(2+) exchange under basal conditions.

Myocardial Function with Reduced Expression of the Sodium-Calcium Exchanger

BACKGROUND The complete removal of the cardiac sodium-calcium exchanger (NCX1) is associated with embryonic lethality, whereas its overexpression is linked to heart failure. To determine whether or not a reduced expression of NCX1 is compatible with normal heart structure and function, we studied 2 knockout (KO) mouse models with reduced levels of NCX1: a heterozygous global KO (HG-KO) with a 50% level of NCX1 expression in all myocytes, and a ventricular-specific KO (V-KO) with NCX1 expression in only 10% to 20% of the myocytes. METHODS AND RESULTS Both groups of mice were evaluated at baseline, after transaortic constriction (TAC), and after acute or chronic beta-adrenergic stimulation. At baseline, the HG-KO mice had smaller hearts and the V-KO mice had larger hearts than their wild-type (WT) controls (P < .05). The HG-KO and their control WT mice had normal responses to TAC and beta-adrenergic stimulation. However, the V-KO group was intolerant to TAC and had a significantly (P < .05) blunted response to beta-adrenergic stimulation as compared with the HG-KO mice and WT controls. Unlike the HG-KO mice, the V-KO mice did not tolerate chronic isoproterenol infusion. Telemetric analysis of the electrocardiogram, body temperature, and activity revealed a normal diurnal rhythm in all groups of mice, but confirmed shorter QT intervals along with increased arrhythmias and reduced R wave to P wave amplitude ratios in the V-KO mice. CONCLUSIONS Though NCX1 can be reduced by half in all myocytes without significant functional alterations, it must be expressed in more than 20% of the myocytes to prevent severe remodeling and heart failure in mouse heart.

Na+/Ca2+ exchanger knockout mice : Plasticity of cardiac excitation-contraction coupling

Abstract:  The Na+/Ca2+ exchanger (NCX) is the main Ca2+ extrusion mechanism of the cardiac myocyte. Nevertheless, cardiac‐specific NCX knockout (KO) mice are viable to adulthood. We have identified two adaptations of excitation–contraction coupling (ECC) to the absence of NCX in these animals: (a) a reduction of the L‐type Ca2+ current (ICa) with an increase in ECC gain and (b) a shortening of the action potential (AP) to further limit Ca2+ influx. Both mechanisms contribute to Ca2+ homeostasis by reducing Ca2+ influx while maintaining contractility. These adaptations may comprise important feedback mechanisms by which cardiomyocytes may be able to limit Ca2+ influx in situations of compromised Ca2+ extrusion capacity.