Molecular Cardiology Research Articles

Upregulation of Myocardial Na+/H+ Exchanger Induced by Chronic Treatment with a Selective Inhibitor

M. C. Camilión de Hurtad, I. L. Ennis, N. G. Pérez, G. E. C.de Cingolani, P. Morgan and H. E. Cingolani. Upregulation of Myocardial Na+/H+ Exchanger Induced by Chronic Treatment with a Selective Inhibitor. Journal of Molecular and Cellular Cardiology (2002) 34, 1539–1547. Rats exposed to prolonged administration of the NHE-1 inhibitor cariporide showed enhanced activity of the exchanger in cardiac tissue, as assessed by the rise in the steady-state pHi value in the absence of bicarbonate (7.15±0.01 in control vs 7.49±0.06 and 7.41±0.05 in cariporide-treated for 1 or 2 months, respectively, P<0.05). In the presence of bicarbonate, the change in pHi was blunted due to a compensatory activation of acid loading pHi regulatory mechanisms. The enhancement of NHE activity disappeared after 1 week of the inhibitor withdrawal. The kinetic analysis of H+ fluxes after an acid load revealed an increased net H+ efflux (JH+) at any given pHi value and an alkaline shift of the apparent “set-point” of the exchanger (from 7.11±0.02 to 7.38±0.04,P <0.05) in treated rats. In the presence of the PKC inhibitor chelerythrine, the “set-point” of the exchanger was normalized in the cariporide-treated rats while JH+ at acidic pHi values persisted elevated. Cardiac NHE-1 mRNA levels and protein expression were increased in cariporide-treated rats. In addition to the increased protein expression after the treatment, the normalization of the augmented “set-point” by chelerythrine suggests an increased turnover rate of the units through a PKC dependent pathway. These data demonstrate that long-term treatment with the NHE-1 inhibitor cariporide enhances the antiporter activity in cardiac tissue through an increase of the number and turnover of functional units. This finding deserves further experimental and clinical evaluations to consider whether it would be advisable a gradual withdrawal of prolonged NHE inhibition to avoid an enhanced response when the exchanger is stimulated.

Excitation-Dependent Intracellular Ca2+ Waves at the Border Zone of the Cryo-injured Rat Heart Revealed by Real-time Confocal Microscopy

H. Tanaka, M. Oyamada, E. Tsujii, T. Nakajo and T. Takamatsu. Excitation-Dependent Intracellular Ca2+ Waves at the Border Zone of the Cryo-injured Rat Heart Revealed by Real-time Confocal Microscopy. Journal of Molecular and Cellular Cardiology (2002) 34, 1501–1512. Intracellular Ca2+ waves, which develop under Ca2+-overloaded conditions of the injured myocardium, are regarded as an important substrate for triggered arrhythmias. However, little is known about whether Ca2+ waves arise or become proarrhythmic in the injured heart in situ. On the hypothesis that injured myocardium manifests frequent Ca2+ waves and produce an oscillatory [Ca2+]i rise leading to triggered activity, we applied cryo-injury to the epicardial surface of fluo 3-AM-loaded perfused rat hearts and analyzed spatiotemporal [Ca2+]i changes at border zones of the injured myocardium using real-time confocal microscopy. In intact regions Ca2+ waves barely emerged, whereas the border zone myocardium exhibited frequent Ca2+ waves, propagating randomly within the individual cells. Two different types of Ca2+ waves were identified: highly frequent waves (159.6±86.5waves/min/cell, n=266) adjacent to the cryo-ablated regions, and less frequent waves (79.0±50.1waves/min/cell, n=160) slightly farther (>2 cells) away from the ablated regions (vicinities). The former Ca2+ waves emerged asynchronously to Ca2+ transients. Contrariwise, the latter depended on ventricular excitation: they vanished instantaneously on Ca2+ transients, but emerged more frequently and propagated more swiftly after cessation of higher-frequency pacing. Immediately after 3-Hz pacing, some cryo-injured hearts exhibited oscillatory [Ca2+]i rises; an instantaneous and synchronous elevation of [Ca2+]i followed by burst occurrence of Ca2+ waves with a gradual decrease in incidence and propagation velocity in a considerable number of cells. These observations indicate that myocardial injury induces Ca2+ waves in the heart, and that their synchronous occurrence could become a substrate for triggered arrhythmias.

Role of the Local Renin–angiotensin System in Cardiac Damage: a Minireview Focussing on Transgenic Animal Models

M. Bader. Role of the Local Renin–angiotensin System in Cardiac Damage: a Minireview Focussing on Transgenic Animal Models. Journal of Molecular and Cellular Cardiology (2002) 34, 1455–1462. The local generation of all components of the renin-angiotensin system (RAS) in the heart has been the basis for the postulation of a tissue RAS in this organ. Since angiotensin II is involved in the induction of cardiac hypertrophy and fibrosis the local generation of this peptide may be of highest clinical importance. Several transgenic animal models have been generated to evaluate the functional importance of the cardiac RAS. We have established a new hypertensive mouse model lacking local angiotensinogen expression in the heart. In these animals, cardiac weight and collagen synthesis are increased compared to normotensive control mice but to a lesser extent than in mice with equally enhanced blood pressure but intact cardiac angiotensinogen generation. Thus, we have shown that local synthesis of this protein is involved but not essential in the development of cardiac hypertrophy and fibrosis.

Gating of the Late Na+ Channel in Normal and Failing Human Myocardium

A. I. Undrovinas, V. A. Maltsev, J., W. Kyle, N. S ilverman and H. N. Sabbah. Gating of the Late Na+ Channel in Normal and Failing Human Myocardium. Journal of Molecular and Cellular Cardiology (2002) 34, 1477–1489. We previously reported an ultraslow inactivating late Na+ current (INaL) in left ventricular cardiomyocytes (VC) isolated from normal (NVC) and failing (FVC) human hearts. This current could play a role in heart failure-induced repolarization abnormalities. To identify properties of NaCh contributing to INaL, we examined early and late openings in cell-attached patches of HEK293 cells expressing human cardiac NaCh α-subunit (α-HEK) and in VC of one normal and three failing human hearts. Two types of the late NaCh openings underlay INaL in all three preparations: scattered late (SLO) and bursts (BO). Amplitude analysis revealed that slope conductance for both SLO and BO was the same compared to the main level of early openings (EO) in both VC (21 vs 22.7pS, NVC; 22.7 vs 22.6pS, FVC) and α-HEK (23.2 vs 23pS), respectively. Analysis of SLO latencies revealed voltage-independent ultraslow inactivation in all preparations with tendency to be slower in FVC compared to NCV. EO and SLO render one open voltage-independent state (τ∼0.4ms) for NVC and FVC. One open (voltage-dependent) and two closed states (one voltage-dependent and another voltage-independent) were found in BO of both specimens. Burst duration tend to be longer in FVC (∼50ms) than in NVC (∼30ms). In FVC we found both modes SLO and BO at membrane potential of −10mV that is attribute for take-off voltages (from −18 to −2mV) for early afterdepolarizations (EAD's) in FVC. In conclusions, we found a novel gating mode SLO that manifest slow (hundreds of ms), voltage-independent inactivation in both NVC and FVC. We were unable to reliably demonstrate any differences in the properties of the late NaCh in failing vs a normal human heart. Accordingly, the late current appears to be generated by a single population of channels in normal and failing human ventricular myocardium. Both SLO and BO could be implicated in EADs in HF.

Glyceraldehyde Phosphate Dehydrogenase Oxidation During Cardiac Ischemia and Reperfusion

P. Eaton, N. Wright, D. J. Hearse and M. J. Shattock. Glyceraldehyde Phosphate Dehydrogenase Oxidation During Cardiac Ischemia and Reperfusion. Journal of Molecular and Cellular Cardiology (2002) 34, 1549–1560.Objectives: Protein S-glutathiolation is a predicted mechanism by which protein thiol groups are oxidized during the oxidative stress of ischaemia and reperfusion. We measured protein S-thiolation during ischaemia and reperfusion and investigated the effect of this oxidative modification on the function of GAPDH.Methods: Glutathione was biotinylated (biotin-GSH) and used to probe for protein S-glutathiolation in isolated rat hearts using non-reducing Western blots and streptavidin-HRP. Streptavidin-agarose was used to purify S-glutathiolated proteins and these were identified using N-terminal sequencing and database searching.Results: Little protein S-glutathiolation occurred in control preparations, but this increased 15-fold during reperfusion. Protein S-glutathiolation was attenuated by the antioxidant mercaptopropionylglycine and was shown to occur only during the firstminutes of reperfusion. Affinity purification of the S-glutathiolated proteins showed 20 dominant S-glutathiolation substrates. A dominant S-thiolated protein was N-terminally sequenced (VKVGVNGFG) and HPLC peptide mapping gave additional sequence nearer the site of oxidation (TGVFTTMEKA). The first sequence was the N-terminus of GAPDH, and the second a peptide from the same protein starting at residue 96. GAPDH was immunopurified from aerobic, ischemic or reperfused hearts. Maleimidofluorescein labeling of purified GAPDH provided an index of its reduced thiol status. In the absence of DTT, ischemia induced a reduction in the number of free thiols on GAPDH that was reversed on reperfusion. When treated with DTT, the free thiol status of GAPDH could be increased in ischemic but not reperfused samples. Ischemia induced a reduction in GAPDH activity that was partially restored by reperfusion. DTT-treatment reactivated ischemic GAPDH, but had little effect on the activity from reperfused tissue. Mass spectra acquired from aerobic GAPDH preparations were relatively simple whereas spectra from ischemic or reperfused preparations were highly complex, possibly indicative of oxidation by multiple oxidants.Conclusions: Many proteins, including GAPDH, are targets for S-glutathiolation during cardiac oxidative stress. GAPDH oxidation is associated with a loss in reduced cysteine status that correlates with the inactivation of this enzyme.

Local Cardiac Renin–Angiotensin System: Hypertension and Cardiac Failure

J. Varagic and E. D. Frohlich. Local Cardiac Renin–Angiotensin System: Hypertension and Cardiac Failure. Journal of Molecular and Cellular Cardiology (2002) 34, 1435–1442. In addition to the effect on arterial pressure, angiotensin II, the effector peptide of the renin–angiotensin system (RAS), exerts mitogenic and growth promoting effects on cardiac myocytes and non-myocytic elements; and both of these effects significantly contribute to the development and progression of hypertensive heart disease (HHD). The traditional concept of the RAS as a systemic, endocrine system has been expanded and the identification of its components in many organs and tissue has been amassed. This paper reviews evidence that supports the concept that the cardiac RAS participate importantly in the development and risk of HHD.

Regulation of Cardiac Fibroblast Cellular Function by Leukemia Inhibitory Factor

F. Wang, J. Trial, J. A. Diwan, F. Gao, H. H. Birdsall, M. L. Entman, P. J. Hornsby, N. Sivasubramaniam and D. L. Mann, Regulation of Cardiac Fibroblast Cellular Function by Leukemia Inhibitory Factor. Journal of Molecular and Cellular Cardiology (2002) 34, 1309–1316. Background: Previous studies have shown that leukemia inhibitory factor (LIF) provokes hypertrophic and cytoprotective effects in cardiac myocytes. However, the effects of LIF in cardiac fibroblasts are not known. Given that the cardiac fibroblast is the most abundant cell type in the heart, we sought to examine the functional effects of LIF on cardiac fibroblasts in vitro. Results: Short-term LIF stimulation (24h) had no effect on fibroblast proliferation and/or cell differentiation. However, longer-term LIF stimulation (48–72h) increased fibroblast proliferation, and significantly inhibited cardiac fibroblast differentiation into myofibroblasts. Moreover, 72h of LIF stimulation significantly reduced collagen content in cardiac fibroblasts cultures, as well as decreased MMP activity in fibroblast culture supernatants. Conclusion: The results of this study suggest that LIF stimulation down-regulates several key components of the remodeling process, including collagen content and matrix metalloproteinase (MMP) activation, and thus suggest that LIF may play an important autocrine/paracrine role in preventing excessive extracellular matrix remodeling following acute myocardial injury.

Physiological Determinants of Contractile Force Generation and Calcium Handling in Mouse Myocardium

<h2>Abstract</h2> L. B. Stull, M. K. Leppo, E. Marbán and P. M. L. Janssen. Physiological Determinants of Contractile Force Generation and Calcium Handling in Mouse Myocardium. <i>Journal of Molecular and Cellular Cardiology</i> (2002) <b>34</b>, 1367–1376. Despite the fact that the mouse has become a common tool to study cardiac dysfunction, little is known regarding the regulation of murine cardiac contractility. We have investigated the three main mechanisms that regulate cardiac output (frequency-dependent activation, length-dependent activation, and β-adrenergic stimulation) in ultra-thin right ventricular (RV) trabeculae from the mouse heart at body temperature (37°C). [Ca²⁺]_i was recorded in a subset of trabeculae iontophoretically loaded with fura-2, and rapid cooling contractures were performed to estimate the sarcoplasmic reticulum (SR) calcium load. The force–frequency relationship was positive (2–12Hz); force increased, albeit slightly, while relaxation timing decreased. As expected, in response to β-adrenergic stimulation, force development increased while contractile duration decreased, and increased muscle length led to increased force generation. Changes in SR calcium load and the calcium transient amplitude paralleled effects on active force generation. Despite several qualitative similarities with other mammalian species, the reserve for augmentation of force via either increased frequency or β-adrenergic stimulation was considerably smaller in mouse than in other animals. Therefore, changes in preload, as opposed to increased HR or adrenergic tone, appears to be a much more important determinant of cardiac performance in the mouse than in larger mammals.

Advanced Glycation Endproduct-Induced Calcium Handling Impairment in Mouse Cardiac Myocytes

R. Petrova, Y. Yamamoto, K. Muraki, H. Yonekura, S. S akurai, T. Watanabe, H. LI, M. Takeuchi, Z. Makita, I. Kato, S. T akasawa, H. Okamoto, Y. Imaizumi and H. Yamamoto. Advanced Glycation Endproduct-Induced Calcium Handling Impairment in Mouse Cardiac Myocytes. Journal of Molecular and Cellular Cardiology (2002) 34, 1425–1431. Long-standing diabetes causes cardiovascular complications including direct cardiac muscle weakening known as diabetic cardiomyopathy. This is characterized by disturbances in both cardiac contraction and relaxation, which are maintained by calcium homeostasis in cardiac cells. Our recent in vitro and in vivo studies have shown that advanced glycation endproducts (AGE) account for diabetic vasculopathy through their engagement of the receptor for AGE (RAGE). Here we show that AGE and RAGE may directly affect the myocardial Ca2+ homeostasis. We created transgenic mice that overexpressed human RAGE in the heart and analyzed the Ca2+ transients in cultivated cardiac myocytes (CM) from the RAGE-transgenic and non-transgenic control fetuses. RAGE overexpression was found to reduce the systolic and diastolic intracellular calcium concentration ([Ca2+]i). Exposure to AGE caused a significant prolongation of the decay time of [Ca2+]i in CM from control mice, and this response was augmented in CM from the RAGE transgenic mice. The results suggest that the AGE and RAGE could play an active role in the development of diabetes-induced cardiac dysfunction.

Expression of Heme Oxygenase-1 in Response to Myocardial Infarction in Rats

P. Lakkisto, E. Palojoki, T. Bäcklund, A. Saraste, I. Tikkanen, L.-M. Voipio-Pulkki and K. Pulkki. Expression of Heme Oxygenase-1 in Response to Myocardial Infarction in Rats. Journal of Molecular and Cellular Cardiology (2002) 34, 1357–1365. Heme oxygenase-1 (HO-1) is a heat shock protein catalysing the degradation of heme to yield biliverdin, carbon monoxide and iron. Several recent studies have proposed the stress-inducible HO-1 to participate in cellular protection also in the heart. We, therefore, examined the expression and localization of HO-1 in a rat experimental myocardial infarction model. Male Wistar rats were subjected to left anterior coronary artery ligation or sham-operation and sacrificed at 1 day, 1 week and 4 weeks after ligation. The expression of HO-1 mRNA was assessed by real-time quantitative RT-PCR and the localization of HO-1 protein by immunoconfocal microscopy. At day 1, HO-1 mRNA was increased 3.9-fold in the peri-infarct border area vs sham-operated hearts (P<0.001) and 2.9-fold vs remote areas of the same hearts (P<0.001). At 1 week, HO-1 mRNA levels remained significantly higher (5-fold) in the peri-infarct border area than in sham-operated hearts (P<0.001). In addition, HO-1 mRNA transiently increased 1.6-fold in the remote non-infarcted myocardium vs sham operated hearts (P<0.05). HO-1 mRNA returned to basal levels by 4 weeks. The increase in HO-1 mRNA was accompanied by increased immunoreactivity of HO-1 protein in the vascular walls throughout the myocardium, and in the cardiomyocytes and fibroblast-like cells of the peri-infarct border areas. Cardiomyocytes showed immunoreactivity at the intercalated disc area, and in the sarcoplasmic reticulum as indicated by the striated pattern of staining. The results suggest that the induction of HO-1 may have an important role in the heart during the first days after myocardial infarction.

Myocardin: A Component of a Molecular Switch for Smooth Muscle Differentiation

J. Chen, C. M. Kitchen, J. W. Streb and J. M. Miano. Myocardin: A Component of a Molecular Switch for Smooth Muscle Differentiation. Journal of Molecular and Cellular Cardiology (2002) 34, 1345–1356. A hallmark of smooth muscle cells (SMCs) in culture and the injured vessel wall is their phenotypic modulation from a differentiated state to one of heightened growth, migration, and matrix synthesis. The transcriptional mechanisms underlying this altered genetic program have yet to be elucidated. Serum response factor (SRF) has emerged as a critical regulator of SMC-restricted gene expression via its interaction with proximal CArG elements; however, levels of SRF protein do not change during SMC phenotypic modulation, suggesting a role for other factors or events in this process. One such factor could be myocardin, a novel SRF coactivator recently cloned from cardiac tissue. Levels of myocardin are abundantly expressed in rat aortic media along with key SMC-restricted genes. In several SMC lines, myocardin mRNA levels decrease in parallel with the loss or attenuation of SMC marker expression. Transient transfection experiments with CMV-driven myocardin in both SMC and non-SMC reveal CArG-dependent transactivation of the SM-Calp promoter-enhancer. Several additional CArG-dependent SMC promoters show variable activation in a cell-and promoter-context dependent manner. To determine whether myocardin could activate an endogenous program of SMC differentiation, we stably transfected L6 myoblasts and assessed SMC marker expression and growth. Results reveal the expression of several SMC markers concomitant with a lower growth potential. Collectively, these studies suggest that myocardin is an important component of a molecular switch for the SMC differentiation program.

Expression of Heme Oxygenase-1 in Response to Myocardial Infarction in Rats

P. Lakkisto, E. Palojoki, T. Bäcklund, A. Saraste, I. Tikkanen, L.-M. Voipio-Pulkki and K. Pulkki. Expression of Heme Oxygenase-1 in Response to Myocardial Infarction in Rats. Journal of Molecular and Cellular Cardiology (2002) 34, 1357–1365. Heme oxygenase-1 (HO-1) is a heat shock protein catalysing the degradation of heme to yield biliverdin, carbon monoxide and iron. Several recent studies have proposed the stress-inducible HO-1 to participate in cellular protection also in the heart. We, therefore, examined the expression and localization of HO-1 in a rat experimental myocardial infarction model. Male Wistar rats were subjected to left anterior coronary artery ligation or sham-operation and sacrificed at 1 day, 1 week and 4 weeks after ligation. The expression of HO-1 mRNA was assessed by real-time quantitative RT-PCR and the localization of HO-1 protein by immunoconfocal microscopy. At day 1, HO-1 mRNA was increased 3.9-fold in the peri-infarct border area vs sham-operated hearts (P<0.001) and 2.9-fold vs remote areas of the same hearts (P<0.001). At 1 week, HO-1 mRNA levels remained significantly higher (5-fold) in the peri-infarct border area than in sham-operated hearts (P<0.001). In addition, HO-1 mRNA transiently increased 1.6-fold in the remote non-infarcted myocardium vs sham operated hearts (P<0.05). HO-1 mRNA returned to basal levels by 4 weeks. The increase in HO-1 mRNA was accompanied by increased immunoreactivity of HO-1 protein in the vascular walls throughout the myocardium, and in the cardiomyocytes and fibroblast-like cells of the peri-infarct border areas. Cardiomyocytes showed immunoreactivity at the intercalated disc area, and in the sarcoplasmic reticulum as indicated by the striated pattern of staining. The results suggest that the induction of HO-1 may have an important role in the heart during the first days after myocardial infarction.

Tissue Angiotensin II During Progression or Ventricular Hypertrophy to Heart Failure in Hypertensive Rats; Differential Effects on PKCε and PKCβ

K. Inagaki, Y. Iwanaga, N. Sarai, Y. Onozawa, H. T akenaka, D. Mochly-Rosen and Y. Kihara. Tissue Angiotensin II During Progression of Ventricular Hypertrophy to Heart Failure in Hypertensive Rats; Differential Effects on PKCε and PKCβ. Journal of Molecular and Cellular Cardiology (2002) 34, 1377–1385. The protein kinase C (PKC) family has been implicated as second messengers in mechanosensitive modulation of cardiac hypertrophy. However, little information is available on the role of expression and activation of specific cardiac PKC isozymes during development of left ventricular hypertrophy (LVH) and failure (LVF). Dahl salt-sensitive rats fed an 8% salt diet developed systemic hypertension and concentric LVH at 11 weeks of age that is followed by left ventricle (LV) dilatation and global hypokinesis at 17 weeks. Among several PKC isozymes expressed in the LV myocardium, only PKCε showed a 94% increase at the LVH stage. At the LVF stage, however, PKCε returned to the control level, whereas PKCβI and βII increased by 158% and 155%, respectively. Hearts were studied at each stage using the Langendorff set-up, and a LV balloon was inflated to achieve an equivalent diastolic wall stress. Following mechanical stretch, PKCε was significantly activated in LVH myocardium in which tissue angiotensin II levels were increased by 59%. Pre-treatment with valsartan, an AT1-receptor blocker, abolished the stretch-mediated PKCε activation. Mechanical stretch no longer induced PKCε activation in LVF. Chronic administration of valsartan blunted the progression of LVF and inhibited the increase in PKCβ. Mechanosensitive PKCε activation is augmented and therefore may contribute to the development of compensatory hypertrophy. This effect was dependent on activation of tissue angiotensin II. However, this compensatory mechanism becomes inactive in LVF, where PKCβ may participate in the progression to cardiac dysfunction and LV remodeling.

Cardioprotection at a Distance: Mesenteric Artery Occlusion Protects the Myocardium via an Opioid Sensitive Mechanism

H. H. Patel, J. Moore, A. K. Hsu and G. J. Gross. Cardioprotection at a Distance: Mesenteric Artery Occlusion Protects the Myocardium via an Opioid Sensitive Mechanism. Journal of Molecular and Cellular Cardiology (2002) 34, 1317–1323. Preconditioning in remote organs protects the myocardium; however, mediators of the protection remain unknown. Protection of the heart is linked to opioids; therefore, we hypothesized that mesenteric preconditioning (MPC) releases endogenous opioids that protect the myocardium from ischemic injury. In an intact rat model of myocardial infarction, all rats underwent 30min of coronary artery occlusion followed by 2h of reperfusion. Prior to coronary artery occlusion, control rats were subjected to sham surgery in which the mesenteric artery was isolated but not occluded both with and without naloxone (10mg/kg) pretreatment. Experimental groups underwent isolation and occlusion of the mesenteric artery for 15min followed by a 10min reperfusion period with and without naloxone pretreatment. At the end of 2h of coronary reperfusion, myocardial infarct size (IS) was determined by tetrazolium staining and expressed as a percent of the area at risk (AAR). Control rats had an IS/AAR of 57.3±2. MPC resulted in a significant reduction in infarct size compared to controls (32.2±3, P<0.001). Pretreatment with naloxone significantly attenuated the protective effects of MPC (53.8±4, P<0.0002). Therefore, it appears that MPC releases endogenous opioids that protect the myocardium from ischemic injury.

Elevated Myocardial Akt Signaling Ameliorates Doxorubicin-induced Congestive Heart Failure and Promotes Heart Growth

Y. Taniyama and K. Walsh. Elevated Myocardial Akt Signaling Ameliorates Doxorubicin-induced Congestive Heart Failure and Promotes Heart Growth. Journal of Molecular and Cellular Cardiology (2002) 34, 1241–1247. Doxorubicin is a chemotherapeutic agent that can induce cardiotoxicity and congestive heart failure (CHF). In this study we tested whether intracoronary Akt1 gene delivery could inhibit doxorubicin-induced CHF. Saline or a replication defective adenoviral vector expressing constitutively-active Akt1 (myrAkt) or β-galactosidase (βgal) was delivered to the myocardium of 8 week old rats one day prior to initiating doxorubicin administration. In animals receiving saline or βgal, doxorubicin resulted in significant decreases in cardiac function and retarded post-natal heart growth at the 5 weeks time point. In contrast, transduction of myrAkt protected hearts against doxorubicin-induced decreases in fractional shortening and cardiac index, and improved left ventricular function at 5 weeks time point. Delivery of myrAkt also reversed the doxorubicin-induced reduction in post-natal heart growth and diminished lung edema. These data show that myocardial Akt can inhibit doxorubicin-induced reductions in cardiac function and growth, suggesting that manipulation of this signaling pathway may have utility for the treatment of congestive heart failure.

The Basic-Helix-Loop-Helix Transcription Factor HAND2 Directly Regulates Transcription of the Atrial Naturetic Peptide Gene

B. D. Thattaliyath, B. A. Firulli and A. B. Firulli. The Basic-Helix-Loop-Helix Transcription Factor HAND2 Directly Regulates Transcription of the Atrial Naturetic Peptide Gene.Journal of Molecular and Cellular Cardiology (2002) 34, 1335–1344. The HAND basic Helix-Loop-Helix (bHLH) transcription factors are essential for the development of heart and extra embryonic structures. Although essential for embryonic development, the molecular pathways in which HAND factors participate are poorly understood. In efforts to identify downstream transcriptional targets, we have determined that HAND2 regulates the transcription of the Atrial Naturetic Peptide (ANP) gene. Results show that ANP expression is reduced in HAND2 null mice. Transactivation assays show significant transcriptional upregulation of ANP by HAND2 and cotransfection experiments using HAND2 and E12 suggest that an E-protein/HAND heterodimer is the likely trans -acting complex. The required cis -elements reside within a 258bp proximal region that contains three evolutionarily conserved Ebox consensus sites. Surprisingly, mutations in these three sites suggest HAND2 activity is DNA-binding independent. In addition, HAND2 and the homeobox factor Nkx2.5 exhibit transcriptional synergy in the regulation of ANP. Taken together, this data shows that HAND2 is an upstream transcriptional regulator of ANP expression, and furthermore HAND2 can synergistically interact with Nkx2.5, showing a functional relationship between HAND2 and Nkx2.5 supporting the genetic observation, that mice null for both HAND2 and Nkx2.5 lack ventricle specification.

Strain-Dependent Variation in Vascular Responses to Nitric Oxide in the Isolated Murine Heart

<h2>Abstract</h2> J. K. Bendall, C. Heymes, T. J. F. Wright, S. Wheatcroft, D. J. Grieve, A. M. Shah and A. C. Cave. Strain-Dependent Variation in Vascular Responses to Nitric Oxide in the Isolated Murine Heart. <i>Journal of Molecular and Cellular Cardiology</i> (2002)<b>34</b> , 1325–1333. Numerous studies in the literature have employed gene-modified mice to investigate vascular function. However, only very limited information exists on baseline murine vascular physiology or on potential variations between different strains. We therefore compared coronary and aortic vascular responses to endothelium-derived vasodilators and exogenous nitric oxide (NO) in three commonly used mouse strains and correlated these data with expression of eNOS, NADPH oxidase subunits, gp91^phox and p67^phox, and superoxide production. Isolated perfused hearts from MF1, 129sv and C57BL/6J mice were subjected to: (a) increasing doses of bradykinin, acetylcholine and sodium nitroprusside, and (b) bolus doses of adenosine and the NO synthase inhibitor, N^G-monomethyl-L -arginine. Vascular responses of thoracic aortic rings were assessed for comparison. Expression of eNOS and NADPH oxidase subunits was assessed by immunoblotting, and superoxide production by lucigenin-enhanced chemiluminescence. Coronary vasodilator responses to bradykinin, acetylcholine and sodium nitroprusside were significantly attenuated in MF1 compared with C57BL/6J and 129sv hearts. Similarly, aortic relaxation to acetylcholine was significantly impaired in MF1 aortic rings compared with in C57BL/6J aortae; these differences were reversed by Tiron. N^G-monomethyl-L -arginine induced significantly less vasoconstriction in MF1 and 129sv hearts compared with C57BL/6J. No differences in aortic relaxation to A23187 or sodium nitroprusside were observed. Cardiac and aortic superoxide production and cardiac expression of p67^phox and gp91^phox were significantly greater in MF1 mice compared with the other strains. There is significant strain-dependent variation in coronary and aortic vascular responsiveness in mice, which may reflect differences in the balance between NO and superoxide generation.

Strain-Dependent Variation in Vascular Responses to Nitric Oxide in the Isolated Murine Heart

J. K. Bendall, C. Heymes, T. J. F. Wright, S. Wheatcroft, D. J. Grieve, A. M. Shah and A. C. Cave. Strain-Dependent Variation in Vascular Responses to Nitric Oxide in the Isolated Murine Heart. Journal of Molecular and Cellular Cardiology (2002)34 , 1325–1333. Numerous studies in the literature have employed gene-modified mice to investigate vascular function. However, only very limited information exists on baseline murine vascular physiology or on potential variations between different strains. We therefore compared coronary and aortic vascular responses to endothelium-derived vasodilators and exogenous nitric oxide (NO) in three commonly used mouse strains and correlated these data with expression of eNOS, NADPH oxidase subunits, gp91phox and p67phox, and superoxide production. Isolated perfused hearts from MF1, 129sv and C57BL/6J mice were subjected to: (a) increasing doses of bradykinin, acetylcholine and sodium nitroprusside, and (b) bolus doses of adenosine and the NO synthase inhibitor, NG-monomethyl-L -arginine. Vascular responses of thoracic aortic rings were assessed for comparison. Expression of eNOS and NADPH oxidase subunits was assessed by immunoblotting, and superoxide production by lucigenin-enhanced chemiluminescence. Coronary vasodilator responses to bradykinin, acetylcholine and sodium nitroprusside were significantly attenuated in MF1 compared with C57BL/6J and 129sv hearts. Similarly, aortic relaxation to acetylcholine was significantly impaired in MF1 aortic rings compared with in C57BL/6J aortae; these differences were reversed by Tiron. NG-monomethyl-L -arginine induced significantly less vasoconstriction in MF1 and 129sv hearts compared with C57BL/6J. No differences in aortic relaxation to A23187 or sodium nitroprusside were observed. Cardiac and aortic superoxide production and cardiac expression of p67phox and gp91phox were significantly greater in MF1 mice compared with the other strains. There is significant strain-dependent variation in coronary and aortic vascular responsiveness in mice, which may reflect differences in the balance between NO and superoxide generation.

Cardiac Energy Metabolism Homeostasis: Role of Cytosolic Calcium

R. S. Balaban. Cardiac Energy Metabolism Homeotasis: Role of Cytosolic Calcium. Journal of Molecular and Cellular Cardiology (2002) 34, 1259–1271. The heart is capable of dramatically altering its overall energy flux with minimal changes in the concentrations of metabolites that are associated with energy metabolism. This cardiac energy metabolism homeostasis is discussed with regard to the potential cytosolic control network responsible for controlling the major energy conversion pathway, oxidative phosphorylation in mitochondria. Several models for this cytosolic control network have been proposed, but a cytosolic Ca2+ dependent parallel activation scheme for metabolism and work is consistent with most of the experimental results. That model proposes that cytosolic Ca2+ regulates both the utilization of ATP by the work producing ATPases as well as the mitochondrial production of ATP. Recent studies have provided evidence supporting this role of cytosolic Ca2+. These data include the demonstration that mitochondrial [Ca2+] can track cytosolic [Ca2+] and that the compartmentation of cytosolic [Ca2+] can facilitate this process. On the metabolic side, Ca2+ has been shown to rapidly activate several steps in oxidative phosphorylation, including F1F0-ATPase ATP production as well as several dehydrogenases, which results in a homeostasis of mitochondrial metabolites similar to that observed in the cytosol. Numerous problems with the Ca2+ parallel activation hypothesis remain including the lack of specific mechanisms of mitochondrial Ca2+ transport and regulation of F1F0-ATPase, the time dependence of Ca2+ activation of cytosolic ATPases as well as oxidative phosphorylation, and the role of cytosolic compartmentation. In addition, the lack of cytosolic or mitochondrial [Ca2+] measurements under in vivo conditions is problematic. Several lines of investigation to address these issues are suggested. A model of the cardiac energy metabolism control network is proposed that includes a Ca2+ parallel activation component together with more classical elements including metabolite feedback and cytosolic compartmentation.

New frontiers in molecular pediatric cardiology.

Numerous advances in understanding the molecular basis of congenital heart disease have been published in the past year. Highlights are reviewed, focusing on two major topics: genetic syndromes and cardiac organogenesis. Genetic syndromes are discussed in the context of complementary data from targeted mutations in animals and genetic mapping studies in humans. These include the DiGeorge, Holt-Oram, Alagille, familial primary pulmonary hypertension, and Noonan syndromes. Novel concepts in cardiac organogenesis are discussed, including the existence and contribution of an anterior heart field to the developing cardiac outflow tract, novel cell-cell signaling involving migrating neural crest, the origins of the conduction system and initial embryonic heartbeat, and the possibility of a population of cardiac stem cells in the adult heart. The studies reviewed have potential clinical relevance in the near future and will be of interest to the clinician interested in congenital heart disease.