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Cardiac sarcomeres produce greater active force in response to stretch, forming the basis of the Frank-Starling mechanism of the heart. The purpose of this study was to provide the systematic understanding of length-dependent activation by investigating experimentally and mathematically how the thin filament 'on--off' switching mechanism is involved in its regulation. Porcine left ventricular muscles were skinned, and force measurements were performed at short (1.9 [micro]m) and long (2.3 [micro]m) sarcomere lengths. We found that 3 mM MgADP increased [Ca.sup.2+] sensitivity of force and the rate of rise of active force, consistent with the increase in thin filament cooperative activation. MgADP attenuated length-dependent activation with and without thin filament reconstitution with the fast skeletal troponin complex (sTn). Conversely, 20 mM of inorganic phosphate (Pi) decreased [Ca.sup.2+] sensitivity of force and the rate of rise of active force, consistent with the decrease in thin filament cooperative activation. Pi enhanced length-dependent activation with and without sTn reconstitution. Linear regression analysis revealed that the magnitude of length-dependent activation was inversely correlated with the rate of rise of active force. These results were quantitatively simulated by a model that incorporates the [Ca.sup.2+]-dependent on-off switching of the thin filament state and interfilament lattice spacing modulation. Our model analysis revealed that the cooperativity of the thin filament on--off switching, but not the [Ca.sup.2+]-binding ability, determines the magnitude of the Frank- Starling effect. These findings demonstrate that the Frank-Starling relation is strongly influenced by thin filament cooperative activation. doi/ 10.1085/jgp.201010502

Nonreceptor tyrosine kinases have an increasingly appreciated role in cardiac injury and protection. To investigate novel tasks for members of the Tec family of nonreceptor tyrosine kinases in cardiac phenotype, we examined the behavior of the Tec isoform in myocardial ischemic injury. Ischemia-reperfusion, but not cardiac protective agents, induced altered intracellular localization of Tec, highlighting distinct actions of this protein compared with other isoforms, such as Bmx, in the same model. Tec is abundantly expressed in cardiac myocytes and assumes a diffuse intracellular localization under basal conditions but is recruited to striated structures upon various stimuli, including ATP. To characterize Tec signaling targets in vivo, we performed an exhaustive proteomic analysis of Tec-binding partners. These experiments expand the role of the Tec family in the heart, identifying the Tec isoform as an ischemic injury-induced isoform, and map the subproteome of its interactors in isolated cells. ischemia; tyrosine kinase; proteomics doi: 10.1152/ajpheart.00273.2010.

Muscular dystrophies are often associated with significant cardiac disease that can be the prominent feature associated with gene mutations in sarcoglycan. Cardiac cell death is a main feature of cardiomyopathy in sarcoglycan deficiency and may arise as a cardiomyocyte intrinsic process that remains unclear. Deficiency of [delta]-sarcoglycan ([delta]-SG) induces disruption of the dystrophin-associated glycoprotein complex, a known cause of membrane instability that may explain cardiomyocytes cytosolic [Ca.sup.2+] increase. In this study we assessed the hypothesis that cytosolic [Ca.sup.2+] increase triggers cardiomyocyte death through mitochondrial [Ca.sup.2+] overload and dysfunction in the [delta]-SG-deficient CHF147 hamster. We showed that virtually all isolated CHF147 ventricular myocytes exhibited elevated cytosolic and mitochondrial [Ca.sup.2+] levels by the use of the Fura-2 and Rhod-2 fluorescent probes. Observation of living cells with Mito-Tracker red lead to the conclusion that ~15% of isolated CHF147 cardiomyocytes had disorganized mitochondria. Transmission electron microscope imaging showed mitochondrial swelling associated with crest and membrane disruption. Analysis of the mitochondrial permeability transition pore (MPTP) activity using calcein revealed that mitochondria of CHF147 ventricular cells were twofold leakier than wild types, whereas reactive oxygen species production was unchanged. Bax, Bcl-2, and LC3 expression analysis by Western blot indicated that the intrinsic apoptosis and the cell death associated to autophagy pathways were not significantly activated in CHF147 hearts. Our results lead to conclusion that cardiomyocytes death in [delta]-SG-deficient animals is an intrinsic phenomenon, likely related to [Ca.sup.2+]-induced necrosis. In this process [Ca.sup.2+] overload-induced MPTP activation and mitochondrial disorganization may have an important role. calcium; mitochondria; cardiac muscle; sarcolemmopathy doi: 10.1152/ajpcell.00545.2009.

Motivated by the need for an analytical tool that can be used routinely to analyze data collected from isolated, detergent-skinned cardiac muscle fibers, we developed a mathematical model for representing the force response to step changes in muscle length (i.e., quick stretch and release). Our proposed model is reasonably simple, consisting of only five parameters representing: (1) the rate constant by which length change-induced distortion of elastic elements is dissipated; (2) the stiffness of the muscle fiber; (3) the amplitude of length-mediated recruitment of stiffness elements; (4) the rate constant by which this length-mediated recruitment takes place; and (5) the magnitude of the nonlinear interaction term by which distortion of elastic elements affects the number of recruited stiffness elements. Fitting this model to a family of force recordings representing responses to eight amplitudes of step length change ([+ or -] 2.0% baseline muscle length in 0.5% increments) enabled four things: (1) reproduction of ali the identifiable features seen in a family of force responses to both positive and negative length changes; (2) close fitting of all records from the whole family of these responses with very little residual error; (3) estimation of all five model parameters with a great degree of certainty; and (4) importantly, ready discrimination between cardiac muscle fibers with different contractile regulatory proteins but showing only subtly different contractile function. We recommend this mathematical model as an analytic tool for routine use in studies of cardiac muscle fiber contractile function. Such model-based analysis gives novel insight to the contractile behavior of cardiac muscle fibers, and it is useful for characterizing the mechanistic effects that alterations of cardiac contractile proteins have on cardiac contractile function. doi/ 10.1085/jgp.201010467

Corresponding to the synchronized contraction of the myocardium and rhythmic pumping function of the heart, a single form of cardiac troponin T (cTnT) is present in the adult cardiac muscle of humans and most other vertebrate species. Alternative splicing variants of cTnT are found in failing human hearts and animal dilated cardiomyopathies. Biochemical analyses have shown that these cTnT variants are functional and produce shifted myofilament [Ca.sup.2+] sensitivity. We proposed a hypothesis that the coexistence of two or more functionally distinct TnT variants in the adult ventricular muscle that is normally activated as a syncytium may decrease heart function and cause cardiomyopathy (Huang et al., Am J Physiol Cell Physiol 294: C213-C222, 2008). In the present study, we studied transgenic mouse hearts expressing one or two cTnT variants in addition to normal adult cTnT to investigate whether desynchronized myofilament activation decreases ventricular efficiency. The function of ex vivo working hearts was examined in the absence of systemic neurohumoral influence. The results showed that the transgenic mouse hearts produced lower maximum left ventricular pressure, slower contractile and relaxation velocities, and decreased stroke volume compared with wild-type controls. Ventricular pumping efficiency, calculated by the ejection integral versus total systolic integral and cardiac work versus oxygen consumption, was significantly lower in transgenic mouse hearts and corresponded to the number of cTnT variants present. The results indicated a pathogenic mechanism in which the coexistence of functionally different cTnT variants in cardiac muscle reduces myocardial efficiency due to desynchronized thin filament activation. cardiac function; muscle contraction; cardiomyopathy; heart failure doi: 10.1152/ajpheart.01105.2009.

Trabeculae carneae are the smallest naturally arising collections of linearly arranged myocytes in the heart. They are the preparation of choice for studies of function of intact myocardium in vitro. In vivo, trabeculae are unique in receiving oxygen from two independent sources: the coronary circulation and the surrounding ventricular blood. Because oxygen partial pressure ([PO.sub.2]) in the coronary arterioles is identical in specimens from both ventricles, whereas that ofventricular blood is 2.5-fold higher in the left ventricle than in the right ventricle, trabeculae represent a 'natural laboratory' in which to examine the influence of'extravascular' [PO.sub.2] on the extent of capillarization of myocardial tissue. We exploit this advantage to test four hypotheses. (1) In trabeculae from either ventricle, a peripheral annulus of cells is devoid of capillaries. (2) Hence, sufficiently small trabeculae from either ventricle are totally devoid of capillaries. (3) The capillary-to-myocyte ratios in specimens from either ventricle are identical to those of their respective walls. (4) Capillary-to-myocyte ratios are comparable in specimens from either ventricle, reflecting equivalent energy demands in vivo, driven by identical contractile frequencies and comparable wall stresses. We applied confocal fluorescent imaging to trabeculae in cross section, subsequently using semi-automated se[G.sub.m]entation techniques to distinguish capillaries from myocytes. We quantified the capillary-to- myocyte ratios of trabeculae from both ventricles and compared them to those determined for the ventricular free walls and septum. Quantitative interpretation was furthered by mathematical modeling, using both the classical solution to the diffusion equation for elliptical cross sections, and a novel approach applicable to cross sections of arbitrary shape containing arbitrary disposition of capillaries and non-respiring collagen cords.

Success of human myocardial tissue engineering for cardiac repair has been limited by adverse effects of scaffold materials, necrosis at the tissue core, and poor survival after transplantation due to ischemic injury. Here, we report the development of scaffold-free prevascularized human heart tissue that survives in vivo transplantation and integrates with the host coronary circulation. Human embryonic stem cells (hESCs) were differentiated to cardiomyocytes by using activin A and BMP-4 and then placed into suspension on a rotating orbital shaker to create human cardiac tissue patches. Optimization of patch culture medium significantly increased cardiomyocyte viability in patch centers. These patches, composed only of enriched cardiomyocytes, did not survive to form significant grafts after implantation in vivo. To test the hypothesis that ischemic injury after transplantation would be attenuated by accelerated angiogenesis, we created 'second-generation,' pre-vascularized, and entirely human patches from cardiomyocytes, endothelial cells (both human umbilical vein and hESC-derived endothelial cells), and fibroblasts. Functionally, vascularized patches actively contracted, could be electrically paced, and exhibited passive mechanics more similar to myocardium than patches comprising only cardiomyocytes. Implantation of these patches resulted in 10-fold larger cell grafts compared with patches composed only of cardiomyocytes. Moreover, the preformed human microvessels anastomosed with the rat host coronary circulation and delivered blood to the grafts. Thus, inclusion of vascular and stromal elements enhanced the in vitro performance of engineered human myocardium and markedly improved viability after transplantation. These studies demonstrate the importance of including vascular and stromal elements when designing human tissues for regenerative therapies. angiogenesis | human embryonic stem cells | tissue engineering myocardial infarction | cardiomyocyte

Left ventricular (LV) circumferential strain and rotation have been introduced as clinical markers of myocardial function. This study investigates how regional LV apical rotation and strain can be used in combination to assess function in the infarcted ventricle. In healthy subjects (n = 15) and patients with myocardial infarction (n = 23), LV apical segmental rotation and strain were measured from apical short-axis recordings by speckle tracking echocardiography (STE) and MRI tagging. Infarct extent was determined by late gadolinium enhancement MRI. To investigate mechanisms of changes in strain and rotation, we used a mathematical finite element simulation model of the LV. Mean apical rotation and strain by STE were lower in patients than in healthy subjects (9.0 [+ or -] 4.9 vs. 12.9 [+ or -] 3.5[degrees] and -13.9 [+ or -]10.7 vs. -23.8 [+ or -] 2.3%, respectively, P < 0.05). In patients, regional strain was reduced in proportion to segmental infarct extent (r = 0.80, P < 0.0001). Regional rotation, however, was similar in the center of the infarct and in remote viable myocardium. Minimum and maximum rotations were found at the infarct borders: minimum rotation at the border zone opposite to the direction of apical rotation, and maximum rotation at the border zone in the direction of rotation. The simulation model reproduced the clinical findings and indicated that the dissociation between rotation and strain was caused by mechanical interactions between infarcted and viable myocardium. Systolic strain reflects regional myocardial function and infarct extent, whereas systolic rotation defines infarct borders in the LV apical region. Regional rotation, however, has limited ability to quantify regional myocardial dysfunction. myocardial infarction; myocardial strain; left ventricular function; left ventricular torsion

The ventricles of small mammals express mostly [alpha]-myosin heavy chain ([alpha]-MHC), a fast isoform, whereas the ventricles of large mammals, including humans, express ~10% [alpha]-MHC on a predominately [beta]-MHC (slow isoform) background. In failing human ventricles, the amount of [alpha]-MHC is dramatically reduced, leading to the hypothesis that even small amounts of [alpha]-MHC on a predominately [beta]-MHC background confer significantly higher rates of force development in healthy ventricles. To test this hypothesis, it is necessary to determine the fundamental rate constants of cross-bridge attachment ([f.sub.app]) and detachment ([g.sub.app]) for myosins composed of 100% [alpha]-MHC or [beta]-MHC, which can then be used to calculate twitch time courses for muscles expressing variable ratios of MHC isoforms. In the present study, rat skinned trabeculae expressing either 100% [alpha]-MHC or 100% [beta]-MHC were used to measure ATPase activity, isometric force, and the rate constant of force redevelopment ([k.sub.tr]) in solutions of varying [Ca.sup.2+] concentrations. The rate of ATP utilization was ~2.5-fold higher in preparations expressing 100% [alpha]-MHC compared with those expressing only [beta]-MHC, whereas [k.sub.tr] was 2-fold faster in the [alpha]-MHC myocardium. From these variables, we calculated [f.sub.app] to be approximately threefold higher for [alpha]-MHC than [beta]-MHC and [g.sub.app] to be twofold higher in [alpha]-MHC. Mathematical modeling of isometric twitches predicted that small increases in [alpha]-MHC significantly increased the rate of force development. These results suggest that low-level expression of [alpha]-MHC has significant effects on contraction kinetics. [alpha]-myosin heavy chain; rate constants of cross-bridge attachment and detachment; rate of rise of force

We performed the present study to determine whether hibernating myocardium is chronically protected from ischemia. Myocardial tissue was rapidly excised from hibernating left anterior descending coronary regions (systolic wall thickening = 2.8 [+ or -] 0.2 vs. 5.4 [+ or -] 0.3 mm in remote myocardium), and high-energy phosphates were quantified by HPLC during simulated ischemia in vitro (37[degrees]C). At baseline, ATP (20.1 [+ or -] 1.0 vs. 26.7 [+ or -] 2.1 [micro]mol/g dry wt, P < 0.05), ADP (8.1 [+ or -] 0.4 vs. 10.3 [+ or -] 0.8 [micro]mol/g, P < 0.05), and total adenine nucleotides (31.2 [+ or -] 1.3 vs. 40.1 [+ or -] 2.9 [micro]mol/g, P < 0.05) were depressed compared with normal myocardium, whereas total creatine, creatine phosphate, and ATP-to-ADP ratios were unchanged. During simulated ischemia, there was a marked attenuation of ATP depletion (5.6 [+ or -] 0.9 vs. 13.7 [+ or -] 1.7 [micro]mol/g at 20 min in control, P < 0.05) and mitochondrial respiration [145 [+ or -] 13 vs. 187 [+ or -] 11 ng atoms [O.sub.2] x mg [protein.sup.-1] x [min.sup.-1] in control (state 3), P < 0.05], whereas lactate accumulation was unaffected. These in vitro changes were accompanied by protection of the hibernating heart from acute stunning during demand-induced ischemia. Thus, despite contractile dysfunction at rest, hibernating myocardium is ischemia tolerant, with reduced mitochondrial respiration and slowing of ATP depletion during simulated ischemia, which may maintain myocyte viability. energy metabolism; mitochondrial respiration; stunned myocardium

Tropomyosin (TM) is involved in [Ca.sup.2+]-mediated muscle contraction and relaxation in the heart. Striated muscle [alpha]-TM is the major isoform expressed in the heart. The expression of striated muscle [beta]-TM in the murine myocardium results in a decreased rate of relaxation and increased myofilament [Ca.sup.2+] sensitivity. Replacing the carboxyl terminus (amino acids 258-284) of [alpha]-TM with [beta]-TM (a troponin T-binding region) results in decreased rates of contraction and relaxation in the heart and decreased myofilament [Ca.sup.2+] sensitivity. We hypothesized that the putative internal troponin T-binding domain (amino acids 175-190) of [beta]-TM may be responsible for the increased myofilament [Ca.sup.2+] sensitivity observed when the entire [beta]-TM is expressed in the heart. To test this hypothesis, we generated transgenic mice that expressed chimeric TM containing [beta]-TM amino acids 175-190 in the backbone of [alpha]-TM (amino acids 1-174 and 191-284). These mice expressed 16-57% chimeric TM and did not develop cardiac hypertrophy or any other morphological changes. Physiological analysis showed that these hearts exhibited decreased rates of contraction and relaxation and a positive response to isoproterenol. Skinned fiber bundle analyses showed a significant increase in myofilament [Ca.sup.2+] sensitivity. Biophysical experiments demonstrated that the exchanged amino acids did not influence the flexibility of the TM. This is the first study to demonstrate that a specific domain within TM can increase the [Ca.sup.2+] sensitivity of the thin filament and affect sarcomeric performance. Furthermore, these results enhance the understanding of why TM mutations associated with familial hypertrophic cardiomyopathy demonstrate increased myofilament sensitivity to [Ca.sup.2+]. calcium sensitivity; contractile function; genetically altered mice

Ectopic activity in cardiac muscle within pulmonary veins (PVs) is associated with the onset and the maintenance of atrial fibrillation in humans. The mechanism underlying this ectopic activity is unknown. Here we investigate automatic activity generated by catecholaminergic stimulation in the rat PV. Intracellular microelectrodes were used to record electrical activity in isolated strips of rat PV and left atrium (LA). The resting cardiac muscle membrane potential was lower in PV [-70 [+ or -] 1 (SE) mV, n = 8] than in LA (-85 [+ or -] 1 mV, n = 8). No spontaneous activity was recorded in PV or LA under basal conditions. Norepinephrine (10-5 M) induced first a hyperpolarization (-8 [+ or -] 1 mV in PV, -3 [+ or -] 1 mV in LA, n = 8 for both) then a slowly developing depolarization (+21 [+ or -] 2 mV after 15 min in PV, +1 [+ or -] 2 mV in LA) of the resting membrane potential. Automatic activity occurred only in PV; it was triggered at approximately -50 mV, and it occurred as repetitive bursts of slow action potentials. The diastolic membrane potential increased during a burst and slowly depolarized between bursts. Automatic activity in the PV was blocked by either atenolol or prazosine, and it could be generated with a mixture of cirazoline and isoprenaline. In both tissues, cirazoline ([10.sup.-6] M) induced a depolarization (+37 [+ or -] 2 mV in PV, n = 5; +5 [+ or -] 1 mV in LA, n = 5), and isoprenaline ([10.sup.-7] M) evoked a hyperpolarization (-11 [+ or -] 3 mV in PV, n = 7; -3 [+ or -] 1 mV in LA, n = 6). The differences in membrane potential and reaction to adrenergic stimulation lead to automatic electrical activity occurring specifically in cardiac muscle in the PV. atrium; norepinephrine; electrophysiology; atrial fibrillation; thoracic veins

Human cytochrome P-450 (CYP)2J2 is abundant in heart and active in biosynthesis of epoxyeicosatrienoic acids (EETs). Recently, we demonstrated that these eicosanoid products protect myocardium from ischemia-reperfusion injury. The present study utilized transgenic (Tr) mice with cardiomyocyte-specific overexpression of human CYP2J2 to investigate protection toward toxicity resulting from acute (0, 5, or 15 mg/kg daily for 3 days, followed by 24-h recovery) or chronic (0, 1.5, or 3.0 mg/kg biweekly for 5 wk, followed by 2-wk recovery) doxorubicin (Dox) administration. Acute treatment resulted in marked elevations of serum lactate dehydrogenase and creatine kinase levels that were significantly greater in wild-type (WT) than CYP2J2 Tr mice. Acute treatment also resulted in less activation of stress response enzymes in CYP2J2 Tr mice (catalase 750% vs. 300% of baseline, caspase-3 235% vs. 165% of baseline in WT vs. CYP2J2 Tr mice). Moreover, CYP2J2 Tr hearts exhibited less Dox-induced cardiomyocytes apoptosis (measured by TUNEL) compared with WT hearts. After chronic treatment, comparable decreases in body weight were observed in WT and CYP2J2 Tr mice. However, cardiac function, assessed by measurement of fractional shortening with M-mode transthoracic echocardiography, was significantly higher in CYP2J2 Tr than WT hearts after chronic Dox treatment (WT 37 [+ or -] 2%, CYP2J2 Tr 47 [+ or -] 1%). WT mice also had larger increases in [beta]-myosin heavy chain and cardiac ankryin repeat protein compared with CYP2J2 Tr mice. CYP2J2 Tr hearts had a significantly higher rate of Dox metabolism than WT hearts (2.2 [+ or -] 0.25 vs. 1.6 [+ or -] 0.50 ng x [min.sup.-1] x 100 [micro]g [protein.sup.-1]). In vitro data from H9c2 cells demonstrated that EETs attenuated Dox-induced mitochondrial damage. Together, these data suggest that cardiac-specific overexpression of CYP2J2 limited Dox-induced toxicity. cytochrome P-450 2J2; heart; function

Cytochrome P-450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce marked reductions in infarct size (IS) in canine myocardium either given before an ischemic insult or at reperfusion similar to that produced in ischemic preconditioning (IPC) and postconditioning (POC) protocols. However, no studies have addressed the possibility that EETs serve a beneficial role in IPC or POC. We tested the hypothesis that EETs may play a role in these two phenomena by preconditioning dog hearts with one 5-min period of total coronary occlusion followed by 10 rain of reperfusion before 60 rain of occlusion and 3 h of reperfusion or by postconditioning with three 30-s periods of reperfusion interspersed with three 30-s periods of occlusion. To test for a role of EETs in IPC and POC, the selective EET antagonists 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) or its derivative, 14,15-epoxyeicosa-5(Z)-enoic acid 2-[2-(3-hydroxypropoxy)-ethoxy]-ethyl ester (14,15-EEZE-PEG), were administered 10 min before IPC, 5 min after IPC, or 5 min before POC. In a separate series, the selective EET synthesis inhibitor N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH) was administered 10 min before IPC. Infarct size was determined by tetrazolium staining and coronary collateral blood flow at 30 min of occlusion and reperfusion flow at 3 h by radioactive microspheres. Both IPC and POC produced nearly equivalent reductions in IS expressed as a percentage of the area at risk (AAR) [Control 21.2 [+ or -] 1.2%, IPC 8.3 [+ or -] 2.2%, POC 10.1 [+ or -] 1.8% (P < 0.001)]. 14,15-EEZE, 14,15-EEZE-PEG, and MS-PPOH markedly attenuated the cardioprotective effects of IPC and POC (14,15-EEZE and 14,15-EEZE-PEG) at doses that had no effect on IS/AAR when given alone. These results suggest a unique role for endogenous EETs in both IPC and POC. cytochrome P-450 epoxygenases; epoxyeicosatrienoic acid antagonists; ischemia-reperfusion

The pregnancy hormone relaxin (RLX) is a powerful cardiostimulatory peptide. Despite its well-characterized effects on the heart, the intracellular mechanisms responsible for RLX's positive inotropic effects are unknown. Cardiac myofilaments are the central contractile elements of the heart, and changes in the phosphorylation status of myofilament proteins are known to mediate changes in function. The first objective of this study was to determine whether RLX stimulates myofilament activation and alters the phosphorylation of one or more myofilament proteins. RLX works through a variety of intracellular signaling cascades in different tissue types. Protein kinases A (PKA) and C (PKC) are two common molecules implicated in RLX signaling and are known to affect myofilament function. Thus the second objective of this study was to determine whether RLX mediates its myocardial effects through PKA or PKC activation. Murine myocardium was treated with recombinant H2-RLX, and cardiac myofilaments were isolated. RLX increased cardiac myofilament force development at physiological levels of intracellular [Ca.sup.2+] without altering myofilament ATP consumption. Myosin binding protein C, troponin T, and troponin I phosphorylation levels were increased with RLX treatment. Immunoblot analysis revealed an increase in myofilament-associated PKC-[delta], decreases in PKC-[alpha] and -[[beta].sub.II], but no effect on PKC-[epsilon]. Inhibition of PKC with chelerythrine chloride or PKC-[delta] with rottlerin prevented the RLX-dependent changes in myofilament function and protein phosphorylation. PKA antagonism with H-89 had no effect on the myofilament effects of RLX. This study is the first to show that RLX-dependent changes in myofilament-associated PKC alters myofilament activation in a manner consistent with its cardiostimulatory effects. heart; ventricular myocardium; hormone; phosphorylation; intracellular signaling

Comparative analysis of the bioenergetic parameters of adult rat cardiomyocytes (CM) and HL-1 cells with very different structure but similar cardiac phenotype was carried out with the aim of revealing the importance of the cell structure for regulation of its energy fluxes. Confocal microscopic analysis showed very different mitochondrial arrangement in these cells. The cytochrome content per milligram of cell protein was decreased in HL-1 cells by a factor of 7 compared with CM. In parallel, the respiratory chain complex activities were decreased by 4-8 times in the HL-1 cells. On the contrary, the activities of glycolytic enzymes, hexokinase (HK), and pyruvate kinase (PK) were increased in HL-1 cells, and these cells effectively transformed glucose into lactate. At the same time, the creatine kinase (CK) activity was significantly decreased in HL-1 cells. In conclusion, the results of this study comply with the assumption that in contrast to CM in which oxidative phosphorylation is a predominant provider of ATP and the CK system is a main carrier of energy from mitochondria to ATPases, in HL-1 cells the energy metabolism is based mostly on the glycolytic reactions coupled to oxidative phosphorylation through HK. Key words: cardiomyocytes, HL-1 cells, respiration, cytochromes, glycolysis, energy metabolism. On a effectue chez des rats adultes une analyse comparee des parametres bioenergetique des cardiomyocytes (CM) et des cellules HL-1, qui ont une structure tres differente mais un phenotype cardiaque similaire, pour montrer l'importance de la structure cellulaire pour la regulation de ses flux energetiques. L'analyse par microscopie confocale a montre une repartition mitochondriale differente dans ces cellules. La teneur en cytochromes par mg de proteine cellulaire a diminue d'un facteur 7 dans les cellules HL-1 par comparaison a celles des CM. Parallelement, les activites complexes de la chaine respiratoire ont diminue de 4 a 8 fois dans les cellules HL-1. En revanche, les activites des enzymes glycolytiques, hexokinase (HK) et pyruvate kinase (PK), ont augmente dans les cellules HL-1, lesquelles ont transforme efficacement le glucose en lactate. L'activite de la creatine kinase (CK) des cellules HL-1 a aussi diminue significativement. En conclusion, les resultats de cette etude viennent etayer a l'hypothese que, contrairement aux CM ou la phosphorylation oxydative est un fournisseur important d'ATP et le systeme CK, le principal transporteur d'energie des mitochondries vers les ATPases, dans les cellules HL-1, le metabolisme energetique repose principalement sur les reactions glycolytiques couplees a la phosphorylation oxydative par l'HK. Mots-cles : cardiomyocytes, cellules HL-1, respiration, cytochromes, glycolyse, metabolisme energetique. [Traduit par la Redaction], Introduction Studies of energy metabolism in muscle cells have led to a conclusion that the metabolic compartmentalization and mechanisms of regulation of oxidative phosphorylation are system-level properties giving rise to [...]

Comprehensive management of patients with chronic ischemic disease is a critically important component of clinical practice. Cardiac myocytes have the potential to adapt to limited flow conditions by adjusting contractile function, reducing metabolism, conserving resources, and preserving myocardial integrity to cope with an oxygen and (or) nutrition shortage. A prime metabolic feature of cardiac myocytes affected by chronic ischemia is the return to a fetal gene pattern with predominance of carbohydrates as the substrate for energy. Structural adaptation with multiple intracellular changes is part of the remodeling process in hibernating myocardium. Transmural heterogeneity, which defines the pattern of injury in ventricular cardiomyocytes and the response to chronic ischemia, is a multifactorial process originating from functional, metabolic, and flow differences in subendocardial and subepicardial regions. Autophagy is typically activated in hibernating myocardium and has been identified as a prosurvival mechanism. Chronic ischemia is associated with changes in the number, size, and distribution of gap junctions and may give rise to conduction disturbances and arrhythmogenesis. Differentiation between viable and nonviable myocardium by assessing sensitivity of inotropic reserve is a crucial diagnostic tool that is correlated with the prognosis and outcome for improved contractility after restoration of blood perfusion in afflicted myocardium.Reliable and accurate diagnosis of ischemic, scar, and viable tissues is critical for recover strategies. Although early surgical reinstitution of blood flow is most effective in restoring physiologic function of the hibernating myocardium, several new approaches offer promising alternatives. Among others, vascular endothelial growth factor and fibroblast growth factor-2 (FGF-2), especially its lo-FGF-2 isoform, have been shown to be effective in rapid neovascularization. Substances such as statins, resveratrol, some hormones, and omega-3 fatty acids can improve recovery effect in chronically underperfused hearts. For patients with drug-refractory ischemia, intramyocardial transplantation of stem cells into predefined areas of the heart can enhance vascularization and have beneficial effects on cardiac function. This review of ischemic injury, its heterogeneity, accurate diagnosis, and newer methods of treatment, shows there is much information and tremendous hope for better management of patients with coronary heart disease. Key words: ischemic injury, myocardial heterogeneity, neovascularization, growth factors. La prise en charge globale des patients atteints d'ischemique chronique est d'une importance capitale en pratique clinique. Les cardiomyocytes ont le potentiel de s'adapter a des conditions d'hypoperfusion, en ajustant leur fonction contractile, en diminuant leur metabolisme, en economisant leurs reserves et en preservant l'integrite myocardique pour pallier le manque d'oxygene et (ou) de nutrition. La principale caracteristique des cardiomyocytes en ischemie chronique est le retour a un profil genique foetal avec predominance de glucides comme substrat energetique. L'adaptation structurale par de multiples modifications intracellulaires fait partie du processus de remodelage du myocarde hibernant. L'heterogeneite transmurale definissant le profil de lesion dans les cardiomyocytes ventriculaires et la reponse a une ischemie chronique est multifactorielle, decoulant des differences fonctionnelles, metaboliques et de debit dans les regions sousendocardiques et sub-epicardiques. L'autophagie est generalement activee dans le myocarde hibernant et a ete identifiee comme un mecanisme de pro-survie. L'ischemie chronique est associee a des modifications du nombre, de la taille et de la distribution de jonctions lacunaires, et pourrait contribuer a l'alteration de la conduction et a l' arythmogenese. La differenciation entre myocarde viable, non viable et sensibilite de la reserve inotrope est un element important du diagnostic, qui est correle avec le pronostic et l'obtention d'une meilleure contractilite apres le retablissement de la perfusion sanguine dans le myocarde lese. Un diagnostic fiable et precis de tissu ischemique, cicatriciel et viable est crucial pour les strategies de recuperation. Le retablissement precoce du debit sanguin par une chirurgie est tres efficace pour restaurer la fonction physiologique du coeur hibernant. Plusieurs approches avant-gardistes visant a ameliorer la recuperation du myocarde lese sont en cours de realisation. Entre autres, le facteur de croissance endotheliale vasculaire et le facteur de croissance des fibroblastes-2, en particulier son isoforme lo-FGF-2, se sont averes tres efficaces comme stimulateurs de la neovascularisation. Les statines, le resveratrol, certaines hormones, les acides gras omega-3 et d'autres substances ont aussi montre un effet recuperateur benefique dans le coeur sous-perfuse de facon chronique. La transplantation intramyocardique de cellules souches dans des regions ciblees chez des patients souffrant d'ischemie pharmacoresistante stimule la vascularisation et a des effets benefiques sur la fonction cardiaque. Les nombreuses informations analysee dans cette synthese relativement a la lesion d'ischemie, a son heterogeneite, a une diagnostic precis et aux nouvelles methodes de traitement suscitent enormement d'espoir pour l'amelioration de la prise en charge des patients souffrant d'insuffisance coronaire. Mots-cles : lesion d'ischemie, heterogeneite myocardique, neovascularisation, facteurs de croissance., [Traduit par la Redaction] Introduction A prolonged imbalance between the supply and demand for myocardial oxygen and nutrition leads to a subacute or chronic state of myocardial ischemia that results [...]

Several clinical studies have shown the beneficial cardiovascular effects of fibrates in patients with diabetes and insulin resistance. The ligands of peroxisome proliferator-activated receptor-[alpha] (PPAR-[alpha]) reduce ischemia-reperfusion injury in nondiabetic animals. We hypothesized that the activation of PPAR-[alpha] would exert cardioprotection in type 2 diabetic Goto-Kakizaki (GK) rats, involving mechanisms related to nitric oxide (NO) production via the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. GK rats and agematched Wistar rats (n [greater than or equal to] 7) were given either 1) the PPAR-[alpha] agonist WY-14643 (WY), 2) dimethyl sulfoxide (DMSO), 3) WY and the NO synthase inhibitor [N.sup.G]-nitro-L-arginine (L-NNA), 4) L-NNA, 5) WY and the PI3K inhibitor wortmannin, or 6) wortmannin alone intravenously before a 35-min period of coronary artery occlusion followed by 2 h of reperfusion. Infarct size (IS), expression of endothelial NO synthase (eNOS), inducible NO synthase, and Akt as well as nitrite/ nitrate were determined. The IS was 75 [+ or -] 3% and 72 [+ or -] 4% of the area at risk in the Wistar and GK DMSO groups, respectively. WY reduced IS to 56 [+ or -] 3% in Wistar (P < 0.05) and to 46 [+ or -] 5% in GK rats (P < 0.001). The addition of either L-NNA or wortmannin reversed the cardioprotective effect of WY in both Wistar (IS, 70 [+ or -] 5% and 65 [+ or -] 5%, respectively) and GK (IS, 66 [+ or -] 4% and 64 [+ or -] 4%, P < 0.05, respectively) rats. The expression of eNOS and eNOS Ser1177 in the ischemic myocardium from both strains was increased after WY. The expression of Akt, Akt Ser473, and Akt Thr308 was also increased in the ischemic myocardium from GK rats following WY. Myocardial nitrite/nitrate levels were reduced in GK rats (P < 0.05). The results suggest that PPAR-[alpha] activation protects the type 2 diabetic rat myocardium against ischemia-reperfusion injury via the activation of the PI3K/Akt and NO pathway. peroxisome proliferator-activated receptor-[alpha] ligand, endothelial function; phosphatidylinositol 3-kinase

The mechanism of exercise-induced benefit and angiogenesis in ischemic heart disease remains poorly defined. This study was designed to investigate the effects of exercise training on the expression of angiogenic factors and angiogenesis in the infarcted myocardium [myocarial infaction (MI)]. Sixty-three male FVB mice were used for study and were divided into subgroups to test the response to exercise: the time-dependent expression of angiogenic factors to exercise training in normal (group 1; n = 12) and infarcted myocardium (group 2; n = 15) and the exerciseinduced angiogenic response in normal and infarcted myocardium (group 3; n = 20) as well as the impact of exercise preconditioning on infarcted myocardium (group 4; n = 26). Exercise training consisted of daily treadmill exercise for 1 h for 3 days. Expression of VEGF and its receptors Flt-1 and Flk-1 was upregulated by exercise training in mice with MI. Exercise-induced VEGF expression in the MI group was higher than that in the sham (control) group. Cell proliferation assessment showed a significantly higher (P < 0.05) number of bromodeoxyuridine-positive cells in post-MI mice in the exercise group as opposed to post-MI mice in the sedentary group. 2,3,5-Triphenyltetrazolium chloride staining revealed a profound difference in the size of MI (18.25 [+ or -] 2.93%) in the exercise group versus the sedentary group (29.26 [+ or -] 7.64%, P = 0.02). Moreover, exercise preconditioning before MI promoted VEGF expression at both mRNA and protein levels. In conclusion, activation of VEGF and its receptors occurs in the infarcted mice heart in response to exercise, which results in decreased infarct size and improved angiogenesis. exercise; angiogenesis; ischemic heart disease; vascular endothelial growth factor

There is limited in vivo data in humans evaluating myocardial fat utilization during increased heart work. This study was done to determine myocardial free fatty acid (FFA) metabolism during rest and atrial pacing, which increases cardiac work without changing arterial substrate concentration. We studied seven healthy men and women (age = 49.7 [+ or -] 3.9 yr, BMI = 23.4 [+ or -] 1.1 kg/[m.sup.2], [Vo.sub.2max] = 35.5 [+ or -] 3.0 ml * [kg.sup.-1] * [min.sup.-1], ejection fraction = 68 [+ or -] 3%). After 3 days of dietary control, coronary sinus, femoral arterial and venous, and peripheral venous catheters were placed. Subjects received [[sup.13C]bicarbonate followed by a continuous infusion of [1-[sup.13]C]palmitate through the end of the study. Arterial and coronary sinus blood sampling and measurements of resting coronary sinus blood flow were made during rest and atrial pacing to 120 beats/min. M[Vo.sub.2] increased (P < 0.05) from rest to atrial pacing. Coronary sinus FFA concentration was significantly lower than arterial through rest and atrial pacing (P = 0.007). Isotopically measured myocardial palmitate uptake increased significantly from rest to atrial pacing (P = 0.03). Approximately one-third of palmitate delivery was extracted by the myocardium during rest and atrial pacing. Myocardial [V.sup.13]C[O.sub.2] production and palmitate oxidation increased significantly from rest (P < 0.01) to atrial pacing. Net glycerol balance was significantly greater than zero during rest (P = 0.04) but not different from zero during atrial pacing (P = 0.13). These data suggest that myocardial lipid uptake and oxidation increase with greater heart work during atrial pacing, with a similar relative proportion of fat oxidation to total myocardial energy expenditure. free fatty acid; stable isotopes; myocardial substrate utilization

The renin-angiotensin system (RAS) has been implicated in a variety of adipose tissue functions, including tissue growth, differentiation, metabolism, and inflammation. Although expression of all components necessary for a locally derived adipose tissue RAS has been demonstrated within adipose tissue, independence of local adipose RAS component concentrations from corresponding plasma RAS fluctuations has not been addressed. To analyze this, we varied in vivo rat plasma concentrations of two RAS components, renin and angiotensinogen (AGT), to determine the influence of their plasma concentrations on adipose and cardiac tissue levels in both perfused (plasma removed) and nonperfused samples. Variation of plasma RAS components was accomplished by four treatment groups: normal, DOCA salt, bilateral nephrectomy, and losartan. Adipose and cardiac tissue AGT concentrations correlated positively with plasma values. Perfusion of adipose tissue decreased AGT concentrations by 11.1%, indicating that adipose tissue AGT was in equilibrium with plasma. Cardiac tissue renin levels positively correlated with plasma renin concentration for all treatments. In contrast, adipose tissue renin levels did not correlate with plasma renin, with the exception of extremely high plasma renin concentrations achieved in the losartan-treated group. These results suggest that adipose tissue may control its own local renin concentration independently of plasma renin as a potential mechanism for maintaining a functional local adipose RAS. renin-angiotensin system; angiotensinogen; myocardium; rat; plasma

Soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids. EETs are formed from arachidonic acid during myocardial ischemia and play a protective role against ischemic cell death. Deletion of sEH has been shown to be protective against myocardial ischemia in the isolated heart preparation. We tested the hypothesis that sEH inactivation by targeted gene deletion or pharmacological inhibition reduces infarct size (I) after regional myocardial ischemia-reperfusion injury in vivo. Male C57BL\6J wildtype or sEH knockout mice were subjected to 40 min of left coronary artery (LCA) occlusion and 2 h of reperfusion. Wild-type mice were injected intraperitoneally with 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE), a sEH inhibitor, 30 min before LCA occlusion or during ischemia 10 min before reperfusion. 14,15-EET, the main substrate for sEH, was administered intravenously 15 min before LCA occlusion or during ischemia 5 min before reperfusion. The EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (EEZE) was given intravenously 15 rain before reperfusion. Area at risk (AAR) and I were assessed using fluorescent microspheres and triphenyltetrazolium chloride, and I was expressed as I/AAR. I was significantly reduced in animals treated with AUDA-BE or 14,15-EET, independent of the time of administration. The cardioprotective effect of AUDA-BE was abolished by the EET antagonist 14,15-EEZE. hrununohistochemistry revealed abundant sEH protein expression in left ventricular tissue. Strategies to increase 14,15-EET, including sEH inactivation, may represent a novel therapeutic approach for cardioprotection against myocardial ischemia-reperfusion injury. 14,15-epoxyeicosatrienoic acids; 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester

Most of the myriad (order of [10.sup.9]) interconnected coronary vessels interact nonlinearly with their embedding contracting myocardium. Their dynamic flow can be simulated based on a nonlinear distributive segmental flow model involving highly nonlinear partial differential equations. Such network flow analysis, although of high accuracy, is computationally excessively complex. On the other hand, a corresponding nonlinear lumped analysis is significantly less demanding since it involves ordinary differential equations. This is, however, at the detriment of accuracy. In the present technical report, a nonlinear lumped representation of coronary segmental flow is presented and tested against predictions of the corresponding distributive analysis. The results suggest that under physiological conditions, the proposed lumped model achieves similar accuracy to the distributive one, yet with considerably higher computational speed.

In cardiac muscle, intracellular [Ca.sup.2+] and [Mg.sup.2+] are potent regulators of calcium release from the sarcoplasmic reticulum (SR). It is well known that the free [[Ca.sup.2+]] in the SR ([[[Ca.sup.2+]].sub.L]) stimulates the [Ca.sup.2+] release channels (ryanodine receptor [RYR]2). However, little is known about the action of luminal [Mg.sup.2+], which has not been regarded as an important regulator of [Ca.sup.2+] release. The effects of luminal [Ca.sup.2+] and [Mg.sup.2+] on sheep RYR2 were measured in lipid bilayers. Cytoplasmic and luminal [Ca.sup.2+] produced a synergistic increase in the opening rate of RYRs. A novel, high affinity inhibition of RYR2 by luminal [Mg.sup.2+] was observed, pointing to an important physiological role tot luminal [Mg.sup.2+] in cardiac muscle. At diastolic [[[Ca.sup.2+]].sub.C], luminal [Mg.sup.2+] inhibition wits voltage independent, with [K.sub.i] = 45 [micro]M at luminal [[Ca.sup.2+]] ([[[Ca.sup.2+]].sub.1]) = 100 [micro]M. Luminal and cytoplasmic [Mg.sup.2+] inhibition was alleviated by increasing [[[Ca.sup.2+]].sub.L] or [[[Ca.sup.2+]].sub.C]. [Ca.sup.2+] and [Mg.sup.2+] on opposite sides of the bilayer exhibited competitive effects on RYRs, indicating that they can compete via the pore for common sites. The data were accurately fitted by a model based on a tetrameric RYR structure with four [Ca.sup.2+]-sensing mechanisms on each subunit: activating luminal L-site (40-[micro]M affinity for [Mg.sup.2+] and [Ca.sup.2+]), cytoplasmic A-site (1.2 [micro]M for [Ca.sup.2+] and 60 [micro]M for [Mg.sup.2+]), inactivating cytoplasmic [I.sub.i]-site (~10 mM for [Ca.sup.2+] and [Mg.sup.2+]), and [I.sub.2]-site ( 1.2 [micro]M for [Ca.sup.2+]). Activation of three or more subunits will cause channel opening. [Mg.sup.2+] inhibition occurs primarily by [Mg.sup.2+] displacing [Ca.sup.2+] from the L- and A-sites, and [Mg.sup.2+] fails to open the channel. The model predicts that under physiological conditions, SR load--dependent [Ca.sup.2+] release (1) is mainly determined by [Ca.sup.2+] displacement of [Mg.sup.2+] from the L-site as SR loading increases, and (2) depends on the properties of both luminal and cytoplasmic activation mechanisms.

Stem cell treatment may positively influence recovery and inflammation after shock by multiple mechanisms, including the paracrine release of protective growth factors. Embryonic stem cells (ESCs) are understudied and may have greater protective power than adult bone marrow stem cells (BMSCs). We hypothesized that ESC paracrine protective mechanisms in the heart (decreased injury by enhanced growth factor-mediated reduction of proinflammatory cytokines) would be superior to the paracrine protective mechanisms of the adult stem cell population in a model of surgically induced global ischemia. Adult Sprague-Dawley rat hearts were isolated and perfused via Langendorff model. Hearts were subjected to 25 min of warm global ischemia and 40 min of reperfusion and were randomly assigned into one of four groups: l) vehicle treated; 2) BMSC or ESC preischemic treatment; 3) BMSC or ESC postischemic treatment; and 4) BMSC- or ESC-conditioned media treatment. Myocardial function was recorded, and hearts were analyzed for expression of tissue cytokines and growth factors (ELISA). Additionally, ESCs and BMSCs in culture were assessed for growth factor production (ELISA). ESC-treated hearts demonstrated significantly greater postischemic recovery of function (left ventricular developed pressure, end-diastolic pressure, and maximal positive and negative values of the first derivative of pressure) than BMSC-treated hearts or controls at end reperfusion. ESC-conditioned media (without cells) also conferred cardioprotection at end reperfusion. ESC-infused hearts demonstrated increased VEGF and IL-10 production compared with BMSC hearts. ESC hearts also exhibited decreased proinflammatory cytokine expression compared with MSC hearts. Moreover, ESCs in cell culture demonstrated greater pluripotency than MSCs. ESC paracrine protective mechanisms in surgical ischemia are superior to those of adult stem cells. bone marrow stem cells; vascular endothelial growth factor; tumor necrosis factor; mesenchymal stem cells

The phosphatidyl-inositol 3-kinase (PI3K) signaling pathway regulates multiple cellular processes including cell survival/apoptosis and growth. In the cardiac context, PI3K[alpha] plays important roles in cardiac growth. We have shown that cardiac PI3K activity is highly regulated during development, with the highest levels found during the fetal-neonatal transition period and the lowest levels in the adult. There is a close relationship between cardiomyocyte proliferation and cardiac P13K activity. In adult transgenic mice, however, the prolonged constitutive activation of PI3K[alpha] in the heart results in hypertrophy. To develop a strategy to allow temporally controlled overexpression of cardiac PI3K[alpha] we engineered a tetracycline (tet) transactivator tet-off controlled transgenic mouse line with a conditional overexpression of a cardiac-specific fusion protein of the SH2 domain of p85 and p110[alpha]. Cardiac PI3K activity and Akt phosphorylation were significantly increased in adult mice after transgene induction following the removal of doxycycline for 2 wk. The heart weight-to-body weight ratio was not changed, and there were no signs of cardiomyopathy. The overexpression of PI3K[alpha] resulted in increased left ventricular (LV) developed pressure and the maximal and minimal positive values of the first derivative of LV pressure, but not heart rate, as assessed in Langendorff hearts. Mice overexpressing PI3K[alpha] also had increases in the levels of [Ca.sup.2+]-regulating proteins, including the L-type [Ca.sup.2+] channels, ryanodine receptors, and sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase 2a. Thus the temporally controlled overexpression of cardiac PI3K[alpha] does not induce hypertrophy or cardiomyopathy but results in increased contractility, probably via the increased expression of multiple [Ca.sup.2]+]-regulating proteins. These distinct phenotypes suggest a fundamental difference between transgenic mice with temporal or prolonged activation of cardiac PI3K[alpha]. Akt; [Ca.sup.2+]-regulating proteins; Langendorff; tet-off; phosphatidylinositol 3-kinase

The matrix metalloproteinases (MMPs) play a pivotal role in adverse left ventricular (LV) myocardial remodeling. The transmembrane protein extracellular MMP inducer (EMMPRIN) causes increased MMP expression in vitro, and elevated levels occur in patients with LV failure. However, the direct consequences of a prolonged increase in the myocardial expression of EMMPRIN in vivo remained unexplored. Cardiac-restricted EMMPRIN expression (EMMPRINexp) was constructed in mice using the full-length human EMMPRIN gene ligated to the myosin heavy chain promoter, which yielded approximately a twofold increase in EMMPRIN compared with that of the age/strain-matched wild-type (WT) mice; EMMPRINexp (n = 27) and WT (n = 33) mice were examined at 3.2 [+ or -] 0.1 or at 13.3 [+ or -] 0.5 mo of age (n = 43 and 26, respectively). LV end-diastolic volume (EDV) was similar in young EMMPRINexp and WT mice (54 [+ or -] 2 vs. 57 [+ or -] 3 [micro]l), but LV ejection fraction (EF) was reduced (51 [+ or -] 1 vs. 57 [+ or -] 1%; P < 0.05). In old EMMPRINexp mice, LV EDV was increased compared with WT mice values (76 [+ or -] 3 vs. 58 [+ or -] 3 [micro]l; P < 0.05) and LV EF was significantly reduced (45 [+ or -] 1 vs. 57 [+ or -] 2%; P < 0.05). In EMMPRINexp old mice, myocardial MMP-2 and membrane type-1 MMP levels were increased by >50% from WT values (P < 0.05) and were accompanied by a twofold higher collagen content (P < 0.05). Persistent myocardial EMMPRINexp in aging mice caused increased levels of both soluble and membrane type MMPs, fibrosis, and was associated with adverse LV remodeling. These findings suggest that EMMPRIN is an upstream signaling pathway that can play a mechanistic role in adverse remodeling within the myocardium. ventricular function

Pim-1 kinase exerts potent cardioprotective effects in the myocardium downstream of AKT, but the participation of Pim-1 in cardiac hypertrophy requires investigation. Cardiac-specific expression of Pim-1 (Pim-WT) or the dominant-negative mutant of Pim-1 (Pim-DN) in transgenic mice together with adenoviral-mediated overexpression of these Pim-1 constructs was used to delineate the role of Pim-1 in hypertrophy. Transgenic overexpression of Pim-1 protects mice from pressure-overload-induced hypertrophy relative to wild-type controls as evidenced by improved hemodynamic function, decreased apoptosis, increases in antihypertrophic proteins, smaller myocyte size, and inhibition of hypertrophic signaling after challenge. Similarly, Pim-1 overexpression in neonatal rat cardiomyocyte cultures inhibits hypertrophy induced by endothelin-1. On the cellular level, hearts of Pim-WT mice show enhanced incorporation of BrdU into myocytes and a hypercellular phenotype compared to wild-type controls after hypertrophic challenge. In comparison, transgenic overexpression of Pim-DN leads to dilated cardiomyopathy characterized by increased apoptosis, fibrosis, and severely depressed cardiac function. Furthermore, overexpression of Pim-DN leads to reduced contractility as evidenced by reduced [Ca.sup.2+] transient amplitude and decreased percentage of cell shortening in isolated myocytes. These data support a pivotal role for Pim-1 in modulation of hypertrophy by impacting responses on molecular, cellular, and organ levels.

The catecholamine norepinephrine is required for fetal survival, but its essential function is unknown. When catecholamine-deficient [tyrosine hydroxylase (Th) null] mouse fetuses die at embryonic day (E)13.5-14.5, they resemble wild-type (wt) fetuses exposed to hypoxia. They exhibit bradycardia (28% reduction in heart rate), thin ventricular myocardium (20% reduction in tissue), epicardial detachment, and death with vascular congestion, hemorrhage, and edema. At E12.5, before the appearance of morphological deficits, catecholamine-deficient fetuses are preferentially killed by experimentally induced hypoxia and have lower tissue [Po.sub.2] levels than wt siblings. By microarray analysis (http://www.ncbi.nlm.nih.gov/geo; accession no. GSE10341), hypoxia-inducible factor-1 target genes are induced to a greater extent in null fetuses than in wt siblings, supporting the notion that mutants experience lower oxygen tension or have an enhanced response to hypoxia. Hypoxia induces a 13-fold increase in plasma norepinephrine levels, which would be expected to increase heart rate, thereby improving oxygen delivery in wt mice. Surprisingly, increasing maternal oxygen (inspired [O.sub.2] 33 or 63%) prevents the effects of catecholamine deficiency, restoring heart rate, myocardial tissue, and survival of Th null fetuses to wt levels. We suggest that norepinephrine mediates fetal survival by maintaining oxygen homeostasis. hypoxia; fetal stress; thin myocardium; bradycardia; norepinephrine

There is strong support for the view that the ventricular myocardium has a laminar organization in which myocytes are grouped into branching layers separated by cleavage planes. However, understanding of the extent and functional implications of this architecture has been limited by the lack of a systematic three-dimensional description of the organization of myocytes and associated perimysial collagen. We imaged myocytes and collagen across the left ventricular wall at high resolution in seven normal rat hearts using extended volume confocal microscopy. We developed novel reconstruction and segmentation techniques necessary for the quantitative analysis of three-dimensional myocyte and perimysial collagen organization. The results confirm that perimysial collagen has an ordered arrangement and that it defines a laminar organization. Perimysial collagen is composed of three distinct forms: extensive meshwork on laminar surfaces, convoluted fibers connecting adjacent layers, and longitudinal cords. While myolaminae are the principal form of structural organization throughout most of the wall, they are not seen in the subepicardium, where perimysial collagen is present only as longitudinal cords. connective tissue; perimysium; myocardium; laminar architecture

Extensive evidence indicates that serum response factor (SRF) regulates muscle-specific gene expression and that myocardin family SRF cofactors are critical for smooth muscle cell differentiation. In a yeast two hybrid screen for novel SRF binding partners expressed in aortic SMC, we identified four and a half LIM domain protein 2 (FHL2) and confirmed this interaction by GST pull-down and coimmunoprecipitation assays. FHL2 also interacted with all three myocardin factors and enhanced myocardin and myocardin-related transcription factor (MRTF)-A-dependent transactivation of smooth muscle [alpha]-actin, SM22, and cardiac atrial natriuretic factor promoters in 10T1/2 cells. The expression of FHL2 increased myocardin and MRTF-A protein levels, and, importantly, this effect was due to an increase in protein stability not due to an increase in myocardin factor mRNA expression. Treatment of cells with proteasome inhibitors MG-132 and lactacystin strongly upregulated endogenous MRTF-A protein levels and resulted in a substantial increase in ubiquitin immunoreactivity in MRTF-A immunoprecipitants. Interestingly, the expression of FHL2 attenuated the effects of RhoA and MRTF-B on promoter activity, perhaps through decreased MRTF-B nuclear localization or decreased SRF-CArG binding. Taken together, these data indicate that myocardin factors are regulated by proteasome-mediated degradation and that FHL2 regulates SRF-dependent transcription by multiple mechanisms, including stabilization of myocardin and MRTF-A. smooth muscle; ubiquitin; restenosis

Hypoxia is necessary for fetal development; however, excess hypoxia is detrimental. Hypoxia has been extensively studied in the near-term fetus, but less is known about earlier fetal effects. The purpose of this study was to determine the window of vulnerability to severe hypoxia, what organ system(s) is most sensitive, and why hypoxic fetuses die. We induced hypoxia by reducing maternal-inspired 02 from 21% to 8%, which decreased fetal tissue oxygenation assessed by pimonidazole binding. The mouse fetus was most vulnerable in midgestation: 24 h of hypoxia killed 89% of embryonic day 13.5 (E13.5) fetuses, but only 5% of Ell.5 and 51% of E17.5 fetuses. Sublethal hypoxia at E12.5 caused growth restriction, reducing fetal weight by 26% and protein by 45%. Hypoxia induced HIF-1 target genes, including vascular endothelial growth factor (Vegf), erythropoietin, glucose transporter-1 and insulin-like growth factor binding protein-1 (Igfbp-1), which has been implicated in human intrauterine growth restriction (IUGR). Hypoxia severely compromised the cardiovascular system. Signs of heart failure, including loss of yolk sac circulation, hemorrhage, and edema, were caused by 18-24 h of hypoxia. Hypoxia induced ventricular dilation and myocardial hypoplasia, decreasing ventricular tissue by 50% and proliferation by 21% in vivo and by 40% in isolated cultured hearts. Epicardial detachment was the first sign of hypoxic damage in the heart, although expression of epicardially derived mitogens, such as FGF2, FGF9, and Wnt9b was not reduced. We propose that hypoxia compromises the fetus through myocardial hypoplasia and reduced heart rate. epicardium; myocardium; hypoxia-inducible transcription factor-I; human intrauterine growth restriction; insulin-like growth factor binding protein-1

Although previous studies report a reduction in myocardial volume during systole, myocardial volume changes during the cardiac cycle have not been quantitatively analyzed with high spatiotemporal resolution. We studied the time course of myocardial volume in the anterior mid-left ventricular (LV) wall of normal canine heart in vivo (n = 14) during atrial or LV pacing using transmurally implanted markers and biplane cineradiography (8 ms/frame). During atrial pacing, there was a significant transmural gradient in maximum volume decrease (4.1, 6.8, and 10.3% at subepi, midwall, and subendo layer, respectively, P = 0.002). The rate of myocardial volume increase during diastole was 4.7 [+ or -] 5.8, 6.8 [+ or -] 6.1, and 10.8 [+ or -] 7.7 ml*[min.sup.-l]*[g.sup.-l], respectively, which is substantially larger than the average myocardial blood flow in the literature measured by the microsphere method (0.7-1.3 ml*[min.sup.-1]*[g.sup.-1]). In the early activated region during LV pacing, myocardial volume began to decrease before the LV pressure upstroke. We conclude that the volume change is greater than would be estimated from the known average transmural blood flow. This implies the existence of blood-filled spaces within the myocardium, which could communicate with the ventricular lumen. Our data in the early activated region also suggest that myocardial volume change is caused not by the intramyocardial tissue pressure but by direct impingement of the contracting myocytes on the microvasculature. myocardial structure; ventricular pacing; mechanical dyssynchrony

G protein-coupled receptors (GPCRs) play an essential role in the regulation of cardiovascular function. Therapeutic modulation of GPCRs has proven to be beneficial in the treatment of human heart disease. Myocardial 'orphan' GPCRs, for which the natural ligand is unknown, represent potential novel therapeutic targets for the treatment of heart disease. Here, we describe the expression pattern, signaling pathways, and possible physiological role of the orphan GPR22. GPR22 mRNA analysis revealed a highly restricted expression pattern, with remarkably abundant and selective expression in the brain and heart of humans and rodents. In the heart, GPR22 mRNA was determined to be expressed in all chambers and was comparable with transcript levels of the [[beta].sub.1]-adrenergic receptor as assessed by Taqman PCR. GPR22 protein expression in cardiac myocytes and coronary arteries was demonstrated in the rat heart by immunohistochemistry. When transfected into HEK-293 cells, GPR22 coupled constitutively to [G.sub.i]/[G.sub.o], resulting in the inhibition of adenyl cyclase. No constitutive coupling to [G.sub.s] or [G.sub.q] was observed. Myocardial mRNA expression of GPR22 was dramatically reduced following aortic banding in mice, suggesting a possible role in response to the stress associated with increased afterload. The absence of detectable GPR22 mRNA expression in the hearts of GPR[22.sup.-/-] mice had no apparent effect on normal heart structure or function; however, these mice displayed increased susceptibility to functional decompensation following aortic banding. Thus, we described, for the first time, the expression pattern and signaling for GPR22 and identified a protective role for GPR22 in response to hemodynamic stress resulting from increased afterload. heart failure; hypertrophy; orphan receptor

Previous studies have shown the beneficial effects of the hepatocyte growth factor (HGF) gene on myocardial perfusion and infarction size but not on the regional strain in relationship to global left ventricular function. A noninvasive magnetic resonance (MR) study was performed to determine the effect of a new HGF gene, VM202, expressing two isoforms of HGF, on regional and global left ventricular function. Pigs (8/group) were divided into three groups: 1) controls without infarction; 2) reperfused, infarcted controls; and 3) infarcted, treated (1 h after reperfusion) with VM202 injected at eight sites. Cine, tagging, and delayed enhancement MR images were acquired at 3 and 50 [+ or -] 3 days after infarction. At 50 days, ejection fraction in infarcted, treated animals increased (38 [+ or -] 1% to 47 [+ or -] 2%, P < 0.01) to the level of controls without infarction (52 [+ or -] 1%, P = 0.16) but decreased in infarcted controls (41 [+ or -] 1% to 37 [+ or -] 1%, P < 0.05). Two-dimensional strain improved in remote, peri-infarcted, and infarcted myocardium. Furthermore, the infarction size was smaller in infarcted, treated animals (7.0 [+ or -] 0.5%) compared with infarcted controls (13.2 [+ or -] 1.6%, P < 0.05). Histopathology showed a lack of hypertrophy in myocytes in peri-infarcted and remote myocardium and the formation of islands/peninsulas of myocytes in infarcted, treated animals but not in infarcted controls. In conclusion, the plasmid HGF gene caused a near complete recovery of ejection fraction and improved the radial and circumferential strain of remote, peri-infarcted, and infarcted regions within 50 days. These beneficial effects may be explained by the combined effects of a speedy and significant infarct resorption and island/peninsulas of hypertrophied myocytes within the infarcted territory but not by compensatory hypertrophy. The combined use of cine and tagging MR imaging provides valuable information on the efficacy of gene therapy. gene therapy; heart failure; infarction; magnetic resonance imaging; remodeling

Multiple factors lead to the development and maintenance of chronic heart failure. Blockade of ErbB-2 or ErbB-4 tyrosine kinase receptor signaling leads to dilated cardiomyopathy. ErbB-1 may protect the heart against stress-induced injury and its ligand; epidermal growth factor (EGF) increases myocardial contractility, whereas heparinbinding EGF is essential for normal cardiac function. However, the role of ErbB-1 in control of cardiac function is not clear. We hypothesized that ErbB-1 is essential for maintaining adult cardiac function. Using the ecdysone-inducible gene expression system, we expressed humanized cardiomyocyte-specific dominant-negative ErbB-1 mutant receptors (hErbB-l-mut) in young adult mice that block endogenous cardiac ErbB-1 signaling. Molecular, morphological, and physiological tests (under anesthesia) were performed. As a result, hErbB-l-mut was expressed selectively in cardiomyocytes leading to the blockade of endogenous ErbB-1 phosphorylation and ErbB-2 transphosphorylation. An increase in left ventricular mass, atrial natriuretic factor expression, and histological changes were indicative of cardiac hypertrophy. Cardiac dilation, numerous cardiac lesions, and the loss of the clear boundary between cardiac fibrils were noted histologically. Early and long-term hErbB-l-rout induction led to a significant decrease in fractional shortening and to significant increases in left ventricular end-systolic diameter and volume. The treatment of adenylyl cyclase activator (forskolin analog) normalized the depressed cardiac function. Resting cardiac function returned to normal after reversing mutant expression. A 4-day survival rate of transverse-aortic constricted hErbB-l-mut mice was only 20% compared with 100% in controls. In conclusion, these observations indicate that the blockade of cardiac ErbB-1 signaling leads to the blockade of ErbB-2 signaling and that together they result in cardiac dysfunction. dilated cardiomyopathy; cardiac function; tyrosine kinase; remodeling

High-resolution (11.7 T) cardiac magnetic resonance imaging (MRI) and histological approaches have been employed in tandem to characterize the secondary damage suffered by the murine myocardium following the initial insult caused by ischemia-reperfusion (I/R). I/R-induced changes in the myocardium were examined in five separate groups at the following time points after I/R: 1 h, day 1, day 3, day 7, and day 14. The infarct volume increased from 1 h to day 1 post-I/R. Over time, the loss of myocardial function was observed to be associated with increased infarct volume and worsened regional wall motion. In the infarct region, I/R caused a decrease in end-systolic thickness coupled with small changes in end-diastolic thickness, leading to massive wall thickening abnormalities. In addition, compromised wall thickening was also observed in left ventricular regions adjacent to the infarct region. A tight correlation ([r.sup.2] = 0.85) between measured MRI and triphenyltetrazolium chloride (TTC) infarct volumes was noted. Our observation that until day 3 post-I/P, the infarct size as measured by TTC staining and MRI was much larger than that of the myocytesilent regions in trichrome- or hematoxylin-eosin-stained sections is consistent with the literature and leads to the conclusion that at such an early phase, the infarct site contains structurally intact myocytes that are functionally compromised. Over time, such affected myocytes were noted to structurally disappear, resulting in consistent infarct sizes obtained from MR/ and TTC as well as trichrome and hematoxylin-eosin analyses on day 7 following I/R. Myocardial remodeling following I/R includes secondary myocyte death followed by the loss of cardiac function over time. redox

Rats of the JCR: LA-cp strain, which are homozygous for the cp gene (cp/cp), are obese, insulin-resistant, and hyperinsulinemic. They exhibit associated micro- and macrovascular disease and end-stage ischemic myocardial lesions and are highly stress sensitive. We subjected male cp/cp rats to pair feeding (providing the rats each day with the amount of food eaten by matched freely fed animals), a procedure that alters the diurnal feeding pattern, leading to a state of intermittent caloric restriction. Effects on insulin, glucose, and lipid metabolism, response to restraint stress, aortic contractile/relaxant response, and myocardial lesion frequency were investigated. Pair-fed young (12-wk-old) cp/cp rats had lower insulin and glucose levels (basal and following restraint), consistent with increased insulin sensitivity, but a greater increase in plasma nonesterified fatty acids in response to restraint. These effects were unrelated to lipolytic rates in adipose tissue but may be related to reduced fatty acid oxidation in skeletal muscle. Older (24-wk-old) pair-fed cp/cp rats had significantly reduced plasma triglyceride levels, improved micro- and macrovascular function, and reduced severity of ischemic myocardial lesions. These changes indicate a significant amelioration of end-stage disease processes in this animal model and the complexity of metabolic/physiological responses in studies involving alterations in food intake. The effects illustrate the sensitivity of the JCR:LA-cp rat, an animal model for the metabolic syndrome and associated cardiovascular disease, to the environmental and experimental milieu. Similar stress-related mechanisms may play a role in metabolically induced cardiovascular disease in susceptible human beings. metabolic syndrome; stress; fatty acids; vascular function; myocardial lesions doi: 10.1152/ajpendo.90257.2008.

The clinical success of stem cell therapy for myocardial repair hinges on a better understanding of cardiac fate mechanisms. We have identified small molecules involved in cardiac fate by screening a chemical library for activators of the signature gene Nkx2.5, using a luciferase knockin bacterial artificial chromosome (BAC) in mouse P19CL6 pluripotent stem cells. We describe a family of sulfonylhydrazone (Shz) small molecules that can trigger cardiac tuRNA and protein expression in a variety of embryonic and adult stem/progenitor cells, including human mobilized peripheral blood mononuclear cells (M-PBMCs). Small-molecule-enhanced M-PBMCs engrafted into the rat heart in proximity to an experimental injury improved cardiac function better than control cells. Recovery of cardiac function correlated with persistence of viable human cells, expressing humanspecific cardiac mRNAs and proteins. Shz small molecules are promising starting points for drugs to promote myocardial repair/regeneration by activating cardiac differentiation in M-PBMCs. cardiogenesis | chemical biology | high-throughput screen | myocardial repair

Heart development is a precisely coordinated process of cellular proliferation, migration, differentiation, and integrated morphogenetic interactions, and therefore it is highly susceptible to developmental anomalies such as the congenital heart disease (CHD). One of the major causes of CHD has been shown to be the mutations in key cardiac transcription factors, including nkx2.5. Here, we report the analysis of zebrafish mutant ftk that showed a progressive heart malformation in the later stages of heart morphogenesis. Our analyses revealed that the cardiac muscle maturation and heart morphogenesis in ftk mutants were impaired because of the disorganization of myofibrils. Notably, we found that the expression of nkx2.5 was down-regulated in the ftk heart despite the normal expression of gata4 and tbx5, suggesting a common mechanism for the occurrence of ftk phenotype and CHD. We identified ftk to be a loss-of-function mutation in a connexin gene, cx36.7/early cardiac connexin (ecx), expressed during early heart development. We further showed by a rescue experiment that Nkx2.5 is the downstream mediator of Ecx-mediated signaling. From these results, we propose that the cardiac connexin Ecx and its downstream signaling are crucial for establishing nkx2.5 expression, which in turn promotes unidirectional, parallel alignment of myofibrils and the subsequent proper heart morphogenesis. cardiomyocyte | congenital heart disease | trabeculation | futka | positional cloning

Repolarization alternans is a harbinger of sudden cardiac death, particularly when it becomes spatially discordant. Alternans, a beat-to-beat alternation in the action potential duration (APD) and intracellular Ca ([Ca.sub.i]), can arise from either tissue heterogeneities or dynamic factors. Distinguishing between these mechanisms in normal cardiac tissue is difficult because of inherent complex three-dimensional tissue heterogeneities. To evaluate repolarization alternans in a simpler two-dimensional cardiac substrate, we optically recorded voltage and/or [Ca.sub.i] in monolayers of cultured neonatal rat ventricular myocytes during rapid pacing, before and after exposure to BAY K 8644 to enhance dynamic factors promoting alternans. Under control conditions (n = 37), rapid pacing caused detectable APD alternans in 81% of monolayers, and [Ca.sub.i] transient alternans in all monolayers, becoming spatially discordant in 62%. After BAY K 8644 (n = 28), conduction velocity restitution became more prominent, and APD and [Ca.sub.i] alternans developed and became spatially discordant in all monolayers, with an increased number of nodal lines separating out-of-phase alternating regions. Nodal lines moved closer to the pacing site with faster pacing rates and changed orientation when the pacing site was moved, as predicted for the dynamically generated, but not heterogeneity-based, alternans. Spatial APD gradients during spatially discordant alternans were sufficiently steep to induce conduction block and reentry. These findings indicate that spatially discordant alternans severe enough to initiate reentry can be readily induced by pacing in two-dimensional cardiac tissue and behaves according to predictions for a predominantly dynamically generated mechanism. calcium cycling; arrhythmias

Despite extensive studies, the fundamental mechanisms responsible for the development and progression of cardiovascular diseases have not yet been fully elucidated. Recent experimental and clinical studies have suggested that reactive oxygen species play a major pathological role. Oxidative stress reduction induced by flavonoids has been regarded by many as the most likely mechanism in the protective effects of these compounds; however, there is an emerging view that flavonoids may also exert modulatory actions on protein kinase and lipid kinase signaling pathways. Quercetin, a major flavonoid present in the human diet, has been widely studied, and its biological properties are consistent with its protective role in the cardiovascular system. However, it remains unknown whether the cardioprotective effects of quercetin may also occur through the modulation of genes involved in cell survival. The main goal of this study was to examine the gene expression profiling of cultured rat primary cardiomyocytes treated with quercetin using DNA microarrays and to relate these data to functional effects. Results showed distinct temporal changes in gene expression induced by quercetin and a strong upregulation of phase 2 enzymes, highlighting quercetin ability to act also with an indirect antioxidant mechanism. flavonoids; phase 2 enzymes; oxidative stress

Pharmacological delivery of nitric oxide (NO) stimulates the cardiac [Na.sup.+]-[K.sup.+] pump. However, effects of NO synthesized by NO synthase (NOS) often differ from the effects of NO delivered pharmacologically. In addition, NOS can become 'uncoupled' and preferentially synthesize [O.sub.2*.sup.-], which often has opposing effects to NO. We tested the hypothesis that NOS-synthesized NO stimulates [Na.sup.+]-[K.sup.+] pump activity, and uncoupling of NOS inhibits it. To image NO, we loaded isolated rabbit cardiac myocytes with 4,5-diaminofluorescein-2 diacetate (DAF-2 DA) and measured fluorescence with confocal microscopy. L-Arginine (L-Arg; 500 [micro]mol/l) increased DAF-2 DA fluorescence by 51% compared with control (n = 8; P < 0.05). We used the whole cell patch-clamp technique to measure electrogenic [Na.sup.+]-[K.sup.+] pump current (Ip). Mean [I.sub.p] of 0.35 [+ or -] 0.03 pA/pF (n = 44) was increased to 0.48 [+ or -] 0.03 pA/pF (n = 7, P < 0.05) by 10 [micro]mol/l L-Arg in pipette solutions. This increase was abolished by NOS inhibition with radicicol or by NO-activated guanylyl cyclase inhibition with IH-[l,2,4]oxadiazole[4,3-a]quinoxalin-l-one. We next examined the effect of uncoupling NOS using paraquat. Paraquat (1 mmol/l) induced a 51% increase in the fluorescence intensity of [O.sub.2*.-]-sensitive dye dihydroethidium compared with control (n = 9; P < 0.05). To examine the functional effects of uncoupling, we measured [I.sub.p] with 100 [micro]mol/1 paraquat included in patch pipette solutions. This decreased [I.sub.p] to 0.28 [+ or -] 0.03 pA/pF (n = 12; P < 0.001). The paraquat-induced pump inhibition was abolished by superoxide dismutase (in pipette solutions). We conclude that NOS-mediated NO synthesis stimulates the [Na.sup.+]-[K.sup.+] pump, whereas uncoupling of NOS causes [O.sub.2*.-]-mediated pump inhibition. myocyte; nitric oxide; superoxide: oxidative stress

It is commonly accepted that L-type [Ca.sup.2+] channel-mediated [Ca.sup.2+]-induced [Ca.sup.2+] release (CICR) is the dominant mode of excitation-contraction (E-C) coupling in the adult mammalian heart and that there is no appreciable CICR in neonates. However, we have observed that cell contraction in the neonatal heart was significantly decreased after sarcoplasmic reticulum (SR) [Ca.sup.2+] depletion with caffeine. Therefore, the present study investigated the developmental changes of CICR in rabbit ventricular myocytes at 3, 10, 20, and 56 days of age. We found that the inhibitory effect of the L-type [Ca.sup.2+] current ([I.sub.Ca]) inhibitor nifedipine (Nif; 15 [micro]M) caused an increasingly larger reduction of [Ca.sup.2+] transients on depolarization in older age groups [from ~15% in 3-day-old (3d) myocytes to ~90% in 56-day-old (56d) myocytes]. The remaining [Ca.sup.2+] transient in the presence of Nif in younger age groups was eliminated by the inhibition of [Na.sup.+]/[Ca.sup.2+] exchanger (NCX) with the subsequent addition of 10 [micro]M KB-R7943 (KB-R). Furthermore, [Ca.sup.2+] transients were significantly reduced in magnitude after the depletion of SR [Ca.sup.2+] with caffeine in all age groups, although the effect was significantly greater in the older age groups (from ~40% in 3d myocytes up to ~70% in 56d myocytes). This SR [Ca.sup.2+]-sensitive [Ca.sup.2+] transient in the earliest developmental stage was insensitive to Nil but was sensitive to the subsequent addition of KB-R, indicating the presence of NCX-mediated CICR that decreased significantly with age (from ~37% in 3d myocytes to ~0.5% in 56d myocytes). In contrast, the/ca-mediated CICR increased significantly with age (from ~10% in 3d myocytes to ~70% in 56d myocytes). The CICR gain as estimated by the integral of the CICR [Ca.sup.2+] transient divided by the integral of its [Ca.sup.2+] transient trigger was smaller when mediated by NCX (~1.0 for 3d myocytes) than when mediated by [I.sub.Ca] (~3.0 for 56d myocytes). We conclude that the lower-efficiency NCX-mediated CICR is a predominant mode of CICR in the earliest developmental stages that gradually decreases as the more efficient L-type [Ca.sup.2+] channel-mediated CICR increases in prominence with ontogeny. excitation-contraction coupling; [Na.sup.+]/[Ca.sup.2+] exchanger; L-type [Ca.sup.2+] channels; sarcoplasmic reticulum

Pathophysiological heterogeneity in cardiac tissue is related to the occurrence of arrhythmias. Of importance are regions of slowed conduction, which have been implicated in the formation of conduction block and reentry. Experimentally, it has been a challenge to produce local heterogeneity in a manner that is both reversible and well controlled. Consequently, we developed a dual-zone superfusion chamber that can dynamically create a small (5 mm) central island of heterogeneity in cultured cardiac cell monolayers. Three different conditions were studied to explore the effect of regionally slowed conduction on wave propagation and reentry: depolarization by elevated extracellular potassium, sodium channel inhibition with lidocaine, and cell-cell decoupling with palmitoleic acid. Using optical mapping of transmembrane voltage, we found that the central region of slowed conduction always served as the core region around which a spiral wave formed and then revolved following a period of rapid pacing. Because of the localized slowing in the core region, we observed experimentally for the first time an S shape of the spiral wave front near its tip. These results indicate that a small region of slowed conduction can play a crucial role in the formation, anchoring, and modulation of reentrant spiral waves. arrhythmia; heterogeneity; cell culture; optical mapping

Sphingosine 1 -phosphate (S1P) is a biologically active lysophospholipid that serves as a key regulator of cellular differentiation and survival. Immune stimuli increase SIP synthesis and secretion by mast cells and platelets, implicating this molecule in tissue responses to injury and inflammation. Binding of S1P to [G.sub.i] protein-coupled receptors activates phosphatidylinositol 3-kinase and Akt in a variety of tissues. To elucidate the mechanisms by which SIP enhances adult cardiac myocyte survival during hypoxia, we used a mouse cell culture system in which [S1P.sub.1] receptors were observed to transduce signals from exogenous S1P, an [S1P.sub.1] receptor antibody with agonist properties, and the pharmacological agents FTY720 and SEW2871. [S1P.sub.1] receptor mRNA and protein were abundantly expressed by adult mouse cardiac myocytes. [S1P.sub.1] [S1P.sub.1] receptor axis enhancement of myocyte survival during hypoxia was abolished by phosphatidylinositol 3-kinase inhibition. [S1P.sub.1] receptor function was closely associated with activation of Akt, inactivation of GSK-3[beta], and reduction of cytochrome c release from heart mitochondria. These observations highlight the importance of [S1P.sub.1] receptors on ventricular myocytes as mediators of inducible resistance against cellular injury during severe hypoxic stress. heart; lysophospholipids; ischemia; cardioprotection; mitochondria

We previously showed that lysozyme (Lzm-S), derived from leukocytes, caused myocardial depression in canine sepsis by binding to the endocardial endothelium to release nitric oxide (NO). NO then diffuses to adjacent myocytes to activate the cGMP pathway. In a canine right ventricular trabecular (RVT) preparation, Lzm-S also decreased the inotropic response to field stimulation (FSR) during which the sympathetic and parasympathetic nerves were simulated to measure the adrenergic response. In the present study, we determined whether the pathway by which Lzm-S decreased FSR was different from the pathway by which Lzm-S reduced steady-state (SS) contraction. Furthermore, we determined whether the decrease in FSR was due to a decrease in sympathetic stimulation or enhanced parasympathetic signaling. In the RVT preparation, we found that the inhibitory effect of Lzm-S on FSR was prevented by NO synthase (NOS) inhibitors. A cGMP inhibitor also blocked the depressant activity of Lzm-S. However, in contrast to the Lzm-S-induced decline in SS contraction, chemical removal of the endocardial endothelium by Triton X-100 to eliminate endothelial NO release did not prevent the decrease in FSR. An inhibitory G protein was involved in the effect of Lzm-S, since FSR could be restored by treatment with pertussis toxin. Atropine prevented the Lzm-S-induced decline in FSR, whereas [[beta].sub.1]- and [[beta].sub.2]-adrenoceptor function was not impaired by Lzm-S. These results indicate that the Lzm-S-induced decrease in FSR results from a nonendothelial release of NO. NO then acts through inhibitory G protein to enhance parasympathetic signaling. adrenergic [beta]-receptors; septic shock; myocardial depression; sympathetic response

Aging is associated with insulin resistance, often attributable to obesity and inactivity. Recent evidence suggests that skeletal muscle insulin resistance in aging is associated with mitochondrial alterations. Whether this is true of the senescent myocardium is unknown. Twelve young (Y, 4 years old) and 12 old (O, 11 years old) dogs, matched for body mass, were instrumented with left-ventricular pressure gauges, aortic and coronary sinus catheters, and flow probes on left circumflex artery. Before surgery, all dogs participated in a 6-wk exercise program. Dogs underwent measurements of hemodynamics and plasma substrates before and during a 2-h hyperinsulinemic-euglycemic clamp to measure whole body and myocardial glucose and nonesterified fatty acid uptake. Following the protocol, myocardial and skeletal samples were obtained to measure components of the insulin-signaling cascade and mitochondrial structure. There was no difference in plasma glucose (Y, 90 [+ or -] 4 mg/dl; O, 87 [+ or -] 4 mg/dl), but old dogs had higher (P < 0.02) nonesterified fatty acids (Y, 384 [+ or -] 48 [micro]mol/1; O, 952 [+ or -] 97 [micro]mo1/1) and plasma insulin (Y, 39 [+ or -] 11 pmol/1; O, 108 [+ or -] 18 pmol/1). Old dogs had impaired total body glucose disposition (Y, 11.5 [+ or -] 1 mg x [kg.sup.-1] x [min.sup.-1]; O, 8.0 [+ or -] 0.5 mg x [kg.sup.-1] x [min.sup.-1]; P < 0.05) and insulin-stimulated myocardial glucose uptake (Y, 3.5 [+ or -] 0.3mg x [min.sup.-1] x [g.sup.-1]; O, 1.8 [+ or -] 0.3 mg x [min.sup.-1] x [g.sup.-1]; P < 0.05). The impaired insulin action was associated with altered insulin signaling and glucose transporter (GLUT4) translocation. There were myocardial mitochondrial structural changes observed in association with decreased expression of uncoupling protein-3. Aging is associated with both whole body and myocardial insulin resistance, independent of obesity and inactivity, but involving altered mitochondrial structure and impaired cellular insulin action. myocardium; mitochondria; uncoupling protein-3

Adrenomedullin (ADM) is an endogenous peptide with favorable hemodynamic effects in vivo. In this study, we characterized the direct functional effects of ADM in isolated preparations from human atria and ventricles. In electrically stimulated human nonfailing right atrial trabeculae, ADM (0.0001-1 [micro]mol/1) increased force of contraction in a concentration-dependent manner, with a maximal increase by 35 [+ or -] 8% (at 1 [micro]mol/1; P < 0.05). The positive inotropic effect was accompanied by a disproportionate increase in calcium transients assessed by aequorin light emission [by 76 [+ or -] 20%; force/light ratio ([DELTA]F/[DELTA]L) 0.58 [+ or -] 0.15]. In contrast, elevation of extracellular calcium (from 2.5 to 3.2 mmol/1) proportionally increased force and aequorin light emission ([DELTA]F/[DELTA]L 1.0 [+ or -] 0.1; P < 0.05 vs. ADM). Consistent with a cAMP-dependent mechanism, ADM (1 [micro]mol/1) increased atrial cAMP levels by 90 [+ or -] 12%, and its inotropic effects could be blocked by the protein kinase A (PKA) inhibitor H-89. ADM also exerted positive inotropic effects in failing atrial myocardium and in nonfailing and failing ventricular myocardium. The inotropic response was significantly weaker in ventricular vs. atrial myocardium and in failing vs. nonfailing myocardium. In conclusion, ADM exerts [Ca.sup.2+] -dependent positive inotropic effects in human atrial and less-pronounced effects in ventricular myocardium. The inotropic effects are related to increased cAMP levels and stimulation of PKA. In heart failure, the responsiveness to ADM is reduced in atria and ventricles. contractility; [Ca.sup.2+] handling; human myocardium

Protease-activated receptor-2 (PAR-2) may have proinflammatory effects in some tissues and protective effects in other tissues. The role of PAR-2 in in vivo myocardial ischemia-reperfusion has not yet been determined. This study tested the hypothesis that PAR-2 activation with the PAR-2 agonist peptide SLIGRL (PAR-2 AP) reduces myocardial infarct size when given at reperfusion in vivo, and this cardioprotection involves the ERK1/2 pathway. Anesthetized rats were randomly assigned to the following groups with 30 min of regional ischemia and 3 h reperfusion: 1) control with saline; 2) vehicle (DMSO); 3) PAR-2 AP, 1 mg/kg given intravenously 5 min before reperfusion; 4) scrambled peptide (SP), 1 mg/kg; 5) the ERK1/2 inhibitor PD-98059 (PD), 0.3 mg/kg given l0 min before reperfusion; 6) the phosphatidylinositol 3-kinase inhibitor LY-294002 (LY), 0.3 mg/kg given 10 min before reperfusion; 7) PD + PAR-2 AP, 0.3 mg/kg PD given 5 min before PAR-2 AP; 8) LY + PAR-2 AP, 0.3 mg/kg LY given 5 min before PAR-2 AP; 9) chelerythrine (Chel) alone, 5 mg/kg given 10 min before reperfusion; and 10) Chel + PAR-2 AP, Chel was given 5 min before PAR-2 AP (10 min before reperfusion). Activation of ERKI/2, ERK5, Akt, and the downstream targets of ERK1/2 [P90 RSK and bcl-xl/bcl-2-associated death promoter (BAD)] was determined by Western blot analysis in separate experiments. PAR-2 AP significantly reduced infarct size compared with control (36 [+ or -] 2% vs. 53 [+ or -] 1%, P < 0.05), and SP had no effect on infarct size (53 [+ or -] 3%). PAR-2 AP significantly increased phosphorylation of ERK1/2, p90RSK, and BAD but not Akt or ERK5. Accordingly, the infarct-size sparing effect of PAR-2 AP was abolished by PD (PAR-2 AP, 36 [+ or -] 2% vs. PD + PAR-2 AP, 50 [+ or -] 1%; P < 0.05) and by Chel (Chel + PAR-2 AP, 58 [+ or -] 2%) but not by LY (PAR-2 AP, 36 [+ or -] 2% vs. LY + PAR-2 AP, 38 [+ or -] 3%; P > 0.05). Therefore, PAR-2 activation is cardioprotective in the in vivo rat heart ischemia-reperfusion model, and this protection involves the ERK1/2 pathway and PKC. reperfusion injury; infarct size; protein kinases