myo-mechanical-analysis-of-isolated-skeletal-muscle

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

Alpha (1)-Adrenozeptorsubtypen vermitteln negativen Inotropie im Myokard von Alpha (1A / C)-Knockout-und Wildtyp-M&#xE4;usen.

Cardiac alpha(1)-adrenoceptors (AR) have two predominant subtypes (alpha(1A)-AR and alpha(1B)-AR) however, their roles in regulating contraction are unclear. We determined the effects of stimulating alpha(1A)-AR (using the subtype-selective agonist A61603) and alpha(1B)-AR (using a gene knockout mouse lacking alpha(1A)-AR) separately, and together (using phenylephrine) on Ca(2+) transients, intracellular pH, and contraction of mouse cardiac trabeculae. Stimulation of alpha(1)-AR subtypes separately or together caused a triphasic contractile response. After a transient ( approximately 3%) force rise (phase 1), force declined markedly (phase 2), then partially recovered (phase 3). In phase 2, the force decline (% of initial) with combined alpha(1A)-AR plus alpha(1B)-AR stimulation (50+/-3%) was more than with separate subtype stimulation (P<0.01), suggesting alpha(1A)-AR and alpha(1B)-AR mediate additive effects during phase 2. Force decline in phase 2 paralleled decreases of Ca(2+) transients that were reduced more with combined vs. separate subtype stimulation. During phase 3 the final force reduction was similar with stimulation of alpha(1A)-AR (20+/-5%), or alpha(1B)-AR (20+/-3%), or both (26+/-4%) suggesting alpha(1A)-AR and alpha(1B)-AR mediate non-additive effects during phase 3. In contrast, Ca(2+) transients recovered fully in phase 3 suggesting reduced force in phase 3 involved decreased myofilament Ca(2+)-sensitivity. Decreased Ca(2+)-sensitivity was not mediated by changes of intracellular pH since this was not affected by alpha(1)-AR stimulation. In contrast to mouse trabeculae, rat trabeculae demonstrated a positive inotropic response to alpha(1)-AR stimulation. In conclusion, for mouse myocardium in vitro both alpha(1)-adrenoceptor subtypes mediate negative inotropy involving decreased Ca(2+) transients and a decreased Ca(2+) sensitivity that does not involve altered intracellular pH.

Alpha(1)-adrenoceptor subtypes mediate negative inotropy in myocardium from alpha(1A/C)-knockout and wild type mice.

Two functional alpha(1)-adrenergic receptor (AR) subtypes (alpha(1A) and alpha(1B)) have been identified in the mouse heart. However, it is unclear whether the third known subtype, alpha(1D)-AR, is also present. To investigate this, we determined whether there were alpha(1)-AR responses in hearts from a novel mouse model lacking alpha(1A)- and alpha(1B)-ARs (double knockout) (ABKO). In Langendorff-perfused hearts, alpha(1)-ARs were stimulated with phenylephrine. For ABKO hearts, phenylephrine reduced left ventricular pressure and coronary flow (to 87 +/- 2% and 86 +/- 4% of initial, respectively, n = 11, P < 0.01). These effects were blocked by prazosin and 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]-8-azaspirol[4,5]decane-7,9-dione] dihydrochloride, suggesting that alpha(1D)-AR-mediated responses were present. In contrast, right ventricular trabeculae from ABKO hearts did not respond to phenylephrine, suggesting that in ABKO perfused hearts, the effects of phenylephrine were not mediated by direct actions on cardiomyocytes. A novel finding was that alpha(1)-AR stimulation caused positive inotropy in the wild-type mouse heart, in contrast to negative inotropy observed in mouse cardiac muscle strips. We conclude that mouse hearts lacking alpha(1A)- and alpha(1B)-ARs retain functional alpha(1)-AR responses involving decreases of coronary flow and ventricular pressure that reflect alpha(1D)-AR-mediated vasoconstriction. Furthermore, alpha(1)-AR inotropic responses depend critically on the experimental conditions.

Alpha 1-adrenergic receptor responses in alpha 1AB-AR knockout mouse hearts suggest the presence of alpha 1D-AR.

Alpha 1-adrenergen Rezeptor alpha Antworten in 1AB-AR-Knockout-Maus Herzen deuten auf das Vorhandensein von Alpha 1D-AR.

We used double-knockout mice (ABKO) lacking both predominant myocardial alpha(1)-adrenergic receptor (AR) subtypes (alpha(1A) and alpha(1B)) to determine if alpha(1)-ARs are required for normal myocardial contraction. Langendorff-perfused ABKO hearts had higher developed pressure than wild type (WT) hearts (123 +/- 3 mmHg n = 22 vs. 103 +/- 3 mmHg, n = 38, P < 0.001). Acutely inhibiting alpha(1)-ARs in WT hearts with prazosin did not increase pressure, suggesting that the increased pressure of ABKO hearts was mediated by long-term trophic effects on contraction rather than direct regulatory effects of alpha(1)-AR removal. Similar to perfused hearts, ABKO ventricular trabeculae had higher submaximal force at 2 mM extracellular [Ca(2+)] than WT (11.4 +/- 1.7 vs. 6.9 +/- 0.6 mN/mm(2), n = 8, P < 0.05); however, the peaks of fura-2 Ca(2+) transients were not different (0.79 +/- 0.11 vs. 0.75 +/- 0.16 microM, n = 10-12, P > 0.05), suggesting ABKO myocardium had increased myofilament Ca(2+)-sensitivity. This conclusion was supported by measuring the Ca(2+)-force relationship using tetanization. Increased myofilament Ca(2+)-sensitivity was not explained by intracellular pH, which did not differ between ABKO and WT (7.41 +/- 0.01 vs. 7.39 +/- 0.02, n = 4-6, P > 0.05; from BCECF fluorescence). However, ABKO displayed impaired troponin I phosphorylation, which may have played a role. In contrast to increased submaximal force, ABKO trabeculae had lower maximal force than WT at high extracellular [Ca(2+)] (29.6 +/- 1.9 vs. 37.6 +/- 1.4 mN/mm(2), n = 7, P < 0.01). However, peak cytosolic [Ca(2+)] was not different (1.13 +/- 0.15 vs. 1.19 +/- 0.04 microM, n = 6-7, P > 0.05), suggesting ABKO myocardium had impaired myofilament function. Finally, ABKO myocardium had decreased responsiveness to beta-AR stimulation. We conclude: alpha(1)-ARs are required for normal myocardial contraction; alpha(1)-ARs mediate long-term trophic effects on contraction; loss of alpha(1)-AR function causes some of the functional abnormalities that are also found in heart failure.

Abnormal myocardial contraction in alpha(1A)- and alpha(1B)-adrenoceptor double-knockout mice.

Abnorme myokardiale Kontraktion im Alpha-(1A) - und alpha (1B)-Adrenozeptor-Doppel-Knockout-M&#xE4;usen.

The cardiac-specific tetracycline-regulated gene expression system (tet-system) is a powerful tool using double-transgenic mice. The cardiac alpha-myosin heavy chain promoter (alphaMHC) drives lifetime expression of a tetracycline-inhibited transcription activator (tTA). Crossing alphaMHC-tTA mice with mice containing a tTA-responsive promoter linked to a target gene yields double-transgenic mice having tetracycline-repressed expression of the target gene in the heart. Using the tet-system, some studies use nontransgenic mice for the control group, whereas others use single-transgenic alphaMHC-tTA mice. However, previous studies found that high-level expression of a modified activator protein caused cardiomyopathy. Therefore, we tested whether cardiac expression of tTA was associated with altered function of alphaMHC-tTA mice compared with wild-type (WT) littermates. We monitored in vivo and in vitro function and gene expression profiles for myocardium from WT and alphaMHC-tTA mice. Compared with WT littermates, alphaMHC-tTA mice had a greater heart-to-body weight ratio (approximately 10%), ventricular dilation, and decreased ejection fraction, suggesting mild cardiomyopathy. In vitro, submaximal contractions were greater compared with WT and were associated with greater myofilament Ca2+ sensitivity. Gene expression profiling revealed that the expression of 153 genes was significantly changed by >20% when comparing alphaMHC-tTA with WT myocardium. These findings demonstrate that introduction of the alphaMHC-tTA construct causes significant effects on myocardial gene expression and major functional abnormalities in vivo and in vitro. For studies using the tet-system, these results suggest caution in the use of controls, since alphaMHC-tTA myocardium differs appreciably from WT. Furthermore, the results raise the possibility that the phenotype conferred by a target gene may be influenced by the modified genetic background of alphaMHC-tTA myocardium.

Cardiac transgenesis with the tetracycline transactivator changes myocardial function and gene expression.

Cardiac Transgenese mit der Tetracyclin-Transaktivator &#xE4;ndert Myokardfunktion und Genexpression.

Matrix metalloproteinase-2 (MMP-2) plays a major role in dysfunctional ventricular remodeling following myocardial injury induced by ischemia/reperfusion and heart failure. To directly assess the role of MMP-2 in the absence of superimposed injury, we generated cardiac-specific, constitutively active MMP-2 transgenic mice.

Cardiac transgenic matrix metalloproteinase-2 expression directly induces impaired contractility.

Matrix Metalloprotease-2 (MMP-2) spielt eine wichtige Rolle in der dysfunktionalen ventrikul&auml;re Umbau nach myokardiale Verletzung induziert durch Isch&auml;mie/Reperfusion und Herz-Ausfall. Um die Rolle der MMP-2 keine &uuml;berlagerten Verletzung direkt bewerten zu k&ouml;nnen, haben wir Herz-spezifische, konstitutiv aktive MMP-2 transgene M&auml;use generiert.

Kardiale transgene Matrix Metalloprotease-2 Ausdruck induziert direkt beeintr&auml;chtigte Kontraktilit&auml;t.

Preconditioning protocols that protect the heart from ischemic injury may aid in the development of new therapies. However, the temporal window of cardioprotection is limited to a few days after the preconditioning stimulus. Here we report a sustained cardioprotected phenotype in mice expressing a tetracycline transactivator (tTA) transcription factor under the control of the alpha-myosin heavy chain (alphaMHC) promoter. alphaMHC-tTA mice were originally designed for tetracycline-regulated gene expression in the heart (Tet system). However, we found that after 45 min of global ischemia at 37 degrees C, left ventricular developed pressure (LVDP) of Langendorff-perfused alphaMHC-tTA mouse hearts rapidly recovered in 5 min to 60% of initial levels, whereas LVDP of wild-type (WT) littermates recovered to only 10% of the initial level. Improved postischemic recovery of function for alphaMHC-tTA hearts was associated with a 50% decrease of infarct size and a significantly smaller release of lactate dehydrogenase to the coronary effluent. Improved postischemic recovery was not attributable to differences in coronary flow that was similar for WT- and alphaMHC-tTA hearts during recovery. Moreover, improved postischemic recovery of alphaMHC-tTA hearts was not abolished by inhibitors of classical cardioprotective effectors (mitochondrial ATP-sensitive K+ channels, PKC, or adenosine receptors), suggesting a novel mechanism. Finally, the tetracycline analog doxycycline, which inhibits binding of tTA to DNA, did not abolish improved recovery for alphaMHC-tTA hearts. The sustained cardioprotected phenotype of alphaMHC-tTA hearts may have implications for developing new therapies to minimize cardiac ischemic injury. Furthermore, investigations of cardioprotection using the Tet system may be aberrantly influenced by sustained preconditioning induced by cardiac transgenesis with tTA.

Sustained preconditioning induced by cardiac transgenesis with the tetracycline transactivator.

Vorkonditionierung Protokolle, die das Herz von isch&auml;mischen Sch&auml;digung sch&uuml;tzen k&ouml;nnte bei der Entwicklung neuer Therapien helfen. Allerdings ist das zeitliche Fenster von Cardioprotection beschr&auml;nkt sich auf wenige Tage nach den Vorkonditionierungszyklus Reiz. Hier berichten wir einen nachhaltiges Cardioprotected-Ph&auml;notyp bei M&auml;usen Transkriptionsfaktor Tetracyclin Transactivator (tTA) unter der Kontrolle des Projekttr&auml;gers Alpha-Myosin heavy Chain (AlphaMHC) zum Ausdruck zu bringen. AlphaMHC-tTA M&auml;use wurden urspr&uuml;nglich f&uuml;r Tetracyclin-regulierten Genexpression im Herzen (Tet-System). Allerdings fanden wir, dass nach 45 min von globalen Isch&auml;mie bei 37 Grad C, linke ventrikul&auml;re entwickelten Druck (Autors) von Langendorff-perfused AlphaMHC-tTA Maus Herzen schnell in 5 min auf 60 % des Ausgangsniveaus, wiederhergestellt, w&auml;hrend Autors der Wildtyp (WT) Wurfgeschwister auf nur 10 % der urspr&uuml;nglichen Ebene wiederhergestellt. Verbesserten postischemic Recovery Funktion f&uuml;r AlphaMHC-tTA Herzen war ein R&uuml;ckgang um 50 % der Infarktgr&ouml;&szlig;e und eine deutlich kleinere Freisetzung von Lactat-Dehydrogenase zu koronaren Abwasser zugeordnet. Verbesserten postischemic Recovery entfielen nicht auf Unterschiede in der koronaren Str&ouml;mung, die w&auml;hrend der Wiederherstellung f&uuml;r WT - und AlphaMHC-tTA Herzen &auml;hnlich war. Dar&uuml;ber hinaus verbesserte postischemic Verwertungvon AlphaMHC-tTA Herzen wurde nicht abgeschafft, durch Inhibitoren der klassischen kardioprotektive Effektoren (mitochondriale ATP-Sensitive K + Kan&auml;le, PKC oder Adenosin-Rezeptoren), einen neuen Mechanismus vorgeschlagen. Schlie&szlig;lich schaffte Tetracyclin analoge Doxycyclin, die Bindung der tTA DNA hemmt, verbesserte Wiederherstellung f&uuml;r AlphaMHC-tTA Herzen nicht ab. Der anhaltende Cardioprotected-Ph&auml;notyp der AlphaMHC-tTA Herzen m&ouml;glicherweise Auswirkungen auf die Entwicklung neuer Therapien um kardiale isch&auml;mische Verletzungen zu minimieren. Dar&uuml;ber hinaus k&ouml;nnen Untersuchungen von Cardioprotection mit dem Tet-System durch anhaltende Vorkonditionierung induziert durch kardiale Transgenese mit tTA abnorm beeinflusst werden.

Nachhaltige Vorkonditionierung induziert durch kardiale Transgenese mit Tetracyclin-Transactivator.

An alpha1-adrenergic receptor (alpha1-AR) antagonist increased heart failure in the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT), but it is unknown whether this adverse result was due to alpha1-AR inhibition or a nonspecific drug effect. We studied cardiac pressure overload in mice with double KO of the 2 main alpha1-AR subtypes in the heart, alpha 1A (Adra1a) and alpha 1B (Adra1b). At 2 weeks after transverse aortic constriction (TAC), KO mouse survival was only 60% of WT, and surviving KO mice had lower ejection fractions and larger end-diastolic volumes than WT mice. Mechanistically, final heart weight and myocyte cross-sectional area were the same after TAC in KO and WT mice. However, KO hearts after TAC had increased interstitial fibrosis, increased apoptosis, and failed induction of the fetal hypertrophic genes. Before TAC, isolated KO myocytes were more susceptible to apoptosis after oxidative and beta-AR stimulation, and beta-ARs were desensitized. Thus, alpha1-AR deletion worsens dilated cardiomyopathy after pressure overload, by multiple mechanisms, indicating that alpha1-signaling is required for cardiac adaptation. These results suggest that the adverse cardiac effects of alpha1-antagonists in clinical trials are due to loss of alpha1-signaling in myocytes, emphasizing concern about clinical use of alpha1-antagonists, and point to a revised perspective on sympathetic activation in heart failure.

Alpha1-adrenergic receptors prevent a maladaptive cardiac response to pressure overload.

Alpha1-adrenergen Rezeptor (alpha1-AR) Gegner erh&ouml;ht Herzinsuffizienz in die Antihypertensive und Lipid-senkende Behandlung zu verhindern Herzinfarkt Trial (ALLHAT), aber es ist nicht bekannt, ob dieses negative Ergebnis aufgrund von alpha1-AR-Hemmung oder eine unspezifische Drogenwirkung war. Wir studierten kardiale Druck &Uuml;berladung bei M&auml;usen mit Doppel KO der 2 wichtigsten alpha1-AR Subtypen im Herzen, alpha-1A (Adra1a) und alpha-1-b (Adra1b). 2 Wochen nach quer Aorten Verengung (TAC) KO Maus z&auml;hlte nur 60 % der WT und KO-M&auml;usen zu &uuml;berleben hatten niedrigere Auswurf Br&uuml;che und gr&ouml;&szlig;ere End-diastolischen Volumes als WT-M&auml;use. Mechanistisch, endg&uuml;ltige Herzen Gewicht und Muskelfaser Querschnittsfl&auml;che waren gleich nach TAC KO und WT-M&auml;usen. KO Herzen nachdem TAC interstitielle Fibrose, erh&ouml;ht hatte erh&ouml;hte Apoptose, und nicht Induktion der fetalen Hypertrophe Gene. Vor TAC, waren isoliert KO Myocytes anf&auml;lliger f&uuml;r Apoptosis nach oxidativer und Beta-AR-Stimulation und Beta-ARs wurden desensibilisiert. So verschlechtert alpha1-AR-L&ouml;schung dilatative Kardiomyopathie nach Druck &Uuml;berlastung durch mehrere Mechanismen, die angibt, dass alpha1-Signalisierung f&uuml;r kardiale Anpassung erforderlich ist. Diese Ergebnisse legen nahe, dass die kardialen Nebenwirkungen alpha1-Antagonisten in klinischen Studien durch den Verlust der alpha1-Signalisierung in Muskelzellen sind, betont Besorgnis &uuml;ber klinische Verwendung von alpha1-Antagonisten, und zeigen Sie auf eine &uuml;berarbeitete Perspektive auf sympathische Aktivierung an Herzversagen.

Alpha1-adrenergen Rezeptoren verhindern maladaptive kardiale Antwort auf Druck-&Uuml;berladung.

The left ventricle (LV) and right ventricle (RV) have differing hemodynamics and embryological origins, but it is unclear whether they are regulated differently. In particular, no previous studies have directly compared the LV versus RV myocardial inotropic responses to alpha(1)-adrenergic receptor (alpha(1)-AR) stimulation. We compared alpha(1)-AR inotropy of cardiac trabeculae from the LV versus RV of adult mouse hearts. As previously reported, for mouse RV trabeculae, alpha(1)-AR stimulation with phenylephrine (PE) caused a triphasic contractile response with overall negative inotropy. In marked contrast, LV trabeculae had an overall positive inotropic response to PE. Stimulation of a single subtype (alpha(1A)-AR) with A-61603 also mediated contrasting LV/RV inotropy, suggesting differential activation of multiple alpha(1)-AR-subtypes was not involved. Contrasting LV/RV alpha(1)-AR inotropy was not abolished by inhibiting protein kinase C, suggesting differential activation of PKC isoforms was not involved. However, contrasting LV/RV alpha(1)-AR inotropic responses did involve different effects on myofilament Ca(2+) sensitivity: submaximal force of skinned trabeculae was increased by PE pretreatment for LV but was decreased by PE for RV. For LV myocardium, alpha(1)-AR-induced net positive inotropy was abolished by the myosin light chain kinase inhibitor ML-9. This study suggests that LV and RV myocardium have fundamentally different inotropic responses to alpha(1)-AR stimulation, involving different effects on myofilament function and myosin light chain phosphorylation.

Contrasting inotropic responses to alpha1-adrenergic receptor stimulation in left versus right ventricular myocardium.

Der linke Ventrikel (LV) und rechten Ventrikel (RV) haben unterschiedliche H&auml;modynamik und embryologischen Herkunft, aber es ist unklar, ob sie unterschiedlich geregelt sind. Insbesondere keine fr&uuml;heren Studien haben direkt gegen&uuml;ber das LV gegen&uuml;ber RV myokardialen Inotrope Antworten zu Alpha (1)-adrenerge Rezeptor (alpha(1)-AR) Stimulierung. Wir verglichen die alpha-1-AR Inotropy der kardialen Trabeculae aus der LV gegen&uuml;ber RV der adulten Maus Herzen. Wie bereits berichtet, f&uuml;r Maus RV Trabeculae verursachte alpha 1-AR-Stimulation mit Phenylephrin (PE) eine Triphasic kontraktilen Antwort mit insgesamt negativen Inotropy. Im Gegensatz hatte LV Trabeculae eine insgesamt positive Inotrope Reaktion auf PE. Stimulation der einzelnen Subtyp (alpha(1A)-AR) mit A-61603 auch vermittelte kontrastierenden LV/RV-Inotropy, was darauf hindeutet, dass differentielle Aktivierung der mehrere alpha-1-AR-Untertypen nicht verwickelt war. Kontrastierenden LV/RV-alpha 1-AR-Inotropy wurde nicht abgeschafft, durch die Hemmung der Proteinkinase C, was darauf hindeutet, dass differentielle Aktivierung der PKC-Isoformen nicht verwickelt war. Jedoch unterschiedliche Auswirkungen auf Myofilament Ca(2+) Empfindlichkeit beinhalten gegens&auml;tzliche LV/RV alpha 1-AR Inotrope Antworten: submaximal Kraft der dunkelh&auml;utige Trabeculae erh&ouml;hte sich um PE-Vorbehandlung f&uuml;r LV aber war sanken PE f&uuml;r RV F&uuml;r LV Myokard induzierte Alpha (1) - AR - Net, die positive Inotropy durch die Myosin-Leichtketten-Kinasehemmer ML-9 abgeschafft wurde. Diese Studie legt nahe, dass LV und RV Myokard grundlegend Inotrope Antworten auf alpha-1-AR-Stimulation, mit unterschiedliche Auswirkungen auf die Myofilament-Funktion und Myosin-Leichtketten-Phosphorylierung haben.

Kontrastierende Inotrope Antworten auf alpha1-adrenergen Rezeptor-Stimulation in linke gegen rechtsventrikul&auml;re Myokard.

Matrix metalloproteinases (MMPs) are central to the development and progression of dysfunctional ventricular remodeling after tissue injury. We studied 6 month old heterozygous mice with cardiac-specific transgenic expression of active MMP-2 (MMP-2 Tg). MMP-2 Tg hearts showed no substantial gross alteration of cardiac phenotype compared to age-matched wild-type littermates. However, buffer perfused MMP-2 Tg hearts subjected to 30 min of global ischemia followed by 30 min of reperfusion had a larger infarct size and greater depression in contractile performance compared to wild-type hearts. Importantly, cardioprotection mediated by ischemic preconditioning (IPC) was completely abolished in MMP-2 Tg hearts, as shown by abnormalities in mitochondrial ultrastructure and impaired respiration, increased lipid peroxidation, cell necrosis and persistently reduced recovery of contractile performance during post-ischemic reperfusion. We conclude that MMP-2 functions not only as a proteolytic enzyme but also as a previously unrecognized active negative regulator of mitochondrial function during superimposed oxidative stress.

Transgenic MMP-2 expression induces latent cardiac mitochondrial dysfunction.

Matrix-Metalloproteinasen (MMPs) sind wesentlich f&uuml;r die Entwicklung und den Verlauf der dysfunktionalen ventrikul&auml;re Umgestaltung nach Gewebeverletzungen. Wir studierten 6 Monate alten heterozygote M&auml;use mit Herz-spezifische transgene Ausdruck des aktiven MMP-2 (MMP-2 Tg). MMP-2 Tg Herzen zeigte keine wesentliche Brutto &Auml;nderung der kardialen Ph&auml;notyp im Vergleich zu Wildtyp-Wurfgeschwister altersbezogenen. Jedoch perfused Puffer MMP-2 Tg Herzen 30 min von globalen Isch&auml;mie, gefolgt von 30 min der Reperfusion ausgesetzt hatte eine gr&ouml;&szlig;ere Infarktgr&ouml;&szlig;e und gr&ouml;&szlig;ere Depression in KONTRAKTILER Leistung im Vergleich zu Wildtyp Herzen. Wichtigsten Cardioprotection von isch&auml;mischen Vorkonditionierung (IPC) vermittelt wurde komplett abgeschafft MMP-2 Tg-Herzen, wie gezeigt durch Anomalien im mitochondrialen Ultrastruktur und beeintr&auml;chtigte Atmung, erh&ouml;hte Lipidperoxidation, Zelle Nekrose und dauerhaft reduzierten Wiederaufnahme der kontraktilen Leistung w&auml;hrend des post-ischemic Reperfusion. Wir schlie&szlig;en, dass MMP-2, nicht nur als eine proteolytische Enzym, sondern auch als zuvor unbekannte aktive negativer Regulator der mitochondrialen Funktion bei &uuml;berlagerten oxidativen Stress Funktionen.

Transgene Ausdruck von MMP-2 induziert latenter kardialer mitochondriale Dysfunktion.

Increased signaling by G(i)-coupled receptors has been implicated in dilated cardiomyopathy. To investigate the mechanisms, we used transgenic mice that develop dilated cardiomyopathy after conditional expression of a cardiac-targeted G(i)-coupled receptor (Ro1). Activation of G(i) signaling by the Ro1 agonist spiradoline caused decreased cellular cAMP levels and bradycardia in Langendorff-perfused hearts. However, acute termination of Ro1 signaling with the antagonist nor-binaltorphimine did not reverse the Ro1-induced contractile dysfunction, indicating that Ro1 cardiomyopathy was not due to acute effects of receptor signaling. Early after initiation of Ro1 expression, there was a 40% reduction in the abundance of the sarcoplasmic reticulum Ca(2+)-ATPase (P < 0.05); thereafter, there was progressive impairment of both Ca(2+) handling and force development assessed with ventricular trabeculae. Six weeks after initiation of Ro1 expression, systolic Ca(2+) concentration was reduced to 0.61 +/- 0.08 vs. 0.91 +/- 0.07 microM for control (n = 6-8; P < 0.05), diastolic Ca(2+) concentration was elevated to 0.41 +/- 0.07 vs. 0.23 +/- 0.06 microM for control (n = 6-8; P < 0.01), and the decline phase of the Ca(2+) transient (time from peak to 50% decline) was slowed to 0.25 +/- 0.02 s vs. 0.13 +/- 0.02 s for control (n = 6-8; P < 0.01). Early after initiation of Ro1 expression, there was a ninefold elevation of matrix metalloproteinase-2 (P < 0.01), which is known to cause myofilament injury. Consistent with this, 6 wk after initiation of Ro1 expression, Ca(2+)-saturated myofilament force in skinned trabeculae was reduced to 21 +/- 2 vs. 38 +/- 0.1 mN/mm(2) for controls (n = 3; P < 0.01). Furthermore, electron micrographs revealed extensive myofilament damage. These findings may have implications for some forms of human heart failure in which increased activity of G(i)-coupled receptors leads to impaired Ca(2+) handling and myofilament injury, contributing to impaired ventricular pump function and heart failure.

Expression of a Gi-coupled receptor in the heart causes impaired Ca2+ handling, myofilament injury, and dilated cardiomyopathy.

Erh&ouml;hte Signalisierung durch G (i)-gekoppelte Rezeptoren hat verwickelt worden, in dilatative Kardiomyopathie. Um die Mechanismen zu untersuchen, verwendeten wir transgene M&auml;use, die dilatative Kardiomyopathie nach bedingten Ausdruck einer Herz-bezogene g (i) entwickeln-Rezeptor (Ro1) gekoppelt. Aktivierung des G(i) Signalisierung durch die Ro1 Agonist Spiradoline verursacht nahm der zellul&auml;ren cAMP Ebenen und Bradykardie in Langendorff-perfused Herzen. Akute Beendigung des Ro1 Signalisierung mit der Antagonist noch-Binaltorphimine jedoch nicht umkehren, die Ro1-induzierten kontraktilen Dysfunktion, darauf hinweist, dass Ro1 Kardiomyopathie nicht aufgrund von akuten Wirkungen Rezeptor zu signalisieren wurde. Fr&uuml;h nach Einleitung eines Ro1 Ausdruck, gab es eine 40 % Verringerung der F&uuml;lle von dem sarkoplasmatischen Retikulum Ca(2+)-ATPase (P &lt; 0,05); Danach gab es progressive Beeintr&auml;chtigung der Ca(2+) Handhabung und Kraft-Entwicklung mit ventrikul&auml;ren Trabeculae beurteilt. Sechs Wochen nach Beginn der Ro1 Ausdruck, systolische Ca(2+) Konzentration auf 0,61 +/-0,08 vs. 0.91 +/-0,07 MicroM f&uuml;r Steuerelement reduziert wurde (n = 6-8; P &lt; 0,05), Diastolische Ca(2+) Konzentration wurde erh&ouml;ht, um 0,41 +/-0,07 vs. 0.23 +/-0,06 MicroM f&uuml;r Kontrolle (n = 6-8; P &lt; 0.01), und die R&uuml;ckgang Phase von den Ca(2+)-Transienten (Zeit vom Gipfel zu 50 % R&uuml;ckgang) bremsten auf 0,25 +/-0.02 s vs. 0.13 +/-0.02 s f&uuml;r Kontrolle (n = 6-8; P &lt; 0,01). Fr&uuml;h nach Einleitung eines Ro1 Ausdruck, gab es eine neunfach H&ouml;he von Matrix-Metalloproteasen-2 (P &lt; 0.01), die bekannt, um Myofilament Verletzungen verursachen. Folgerichtig 6 wk nach Beginn der Ro1 Ausdruck, Ca(2+)-ges&auml;ttigte Myofilament Kraft in hellh&auml;utigen Trabeculae wurde reduziert auf 21 +/-2 vs. 38 +/-0,1 mN/mm(2) f&uuml;r Steuerelemente (n = 3; P &lt; 0,01). Electron Aufnahmen ergab dar&uuml;ber hinaus umfangreiche Myofilament Schaden. Diese Ergebnisse m&ouml;glicherweise Auswirkungen hat f&uuml;r einige Formen der menschlichen Herzinsuffizienz in die erh&ouml;hte Aktivit&auml;t von G (i)-gekoppelte Rezeptoren f&uuml;hrt auf gest&ouml;rte Ca(2+) Handhabung und Myofilament Verletzungen zu gest&ouml;rter linksventrikul&auml;rer Pump-Funktion und Herzinsuffizienz beitragen.

Ausdruck von einem Gi-gekoppelter Rezeptor im Herzen verursacht beeintr&auml;chtigt Ca2 + Handling, Myofilament Verletzungen und dilatative Kardiomyopathie.

A non-invasive method for estimating regional myocardial contractility in vivo would be of great value in the design and evaluation of new surgical and medical strategies to treat and/or prevent infarction-induced heart failure. As a first step towards developing such a method, an explicit finite element (FE) model-based formal optimization of regional myocardial contractility in a sheep with left ventricular (LV) aneurysm was performed using tagged magnetic resonance (MR) images and cardiac catheterization pressures. From the tagged MR images, 3-dimensional (3D) myocardial strains, LV volumes and geometry for the animal-specific 3D FE model of the LV were calculated, while the LV pressures provided physiological loading conditions. Active material parameters (T(max_B) and T(max_R)) in the non-infarcted myocardium adjacent to the aneurysm (borderzone) and in myocardium remote from the aneurysm were estimated by minimizing the errors between FE model-predicted and measured systolic strains and LV volumes using the successive response surface method for optimization. The significant depression in optimized T(max_B) relative to T(max_R) was confirmed by direct ex vivo force measurements from skinned fiber preparations. The optimized values of T(max_B) and T(max_R) were not overly sensitive to the passive material parameters specified. The computation time of less than 5 hours associated with our proposed method for estimating regional myocardial contractility in vivo makes it a potentially very useful clinical tool.

A computationally efficient formal optimization of regional myocardial contractility in a sheep with left ventricular aneurysm.

Eine nicht-invasive Methode zur Einsch&auml;tzung der regionalen myokardialen Kontraktilit&auml;t in-vivo w&auml;re von gro&szlig;em Wert f&uuml;r die Gestaltung und Bewertung neuer Strategien f&uuml;r chirurgische und medizinische Behandlungsmethoden und/oder Infarkt-induzierte Herzinsuffizienz verhindern. Als ersten Schritt zur Entwicklung eine solche Methode war eine explizite finite Elemente (FE) Modell-basierte formale Optimierung der regionale myokardiale Kontraktilit&auml;t in ein Schaf mit linken Ventrikel (LV)-Aneurysma mit gekennzeichneten Magnetresonanz (MR) Bilder und kardiale Katheterisierung Druck ausgef&uuml;hrt. Von den gekennzeichneten Herr Bildern wurden dreidimensionale (3D) myokardialen St&auml;mme, LV B&auml;nde und Geometrie f&uuml;r Tier-spezifische FE 3D-Modell des LV berechnet, w&auml;hrend der LV-Druck physiologische Ladebedingungen zur Verf&uuml;gung gestellt. Aktive Materialparameter (T(max_B) und T(max_R)) im nicht-infarcted Myokard angrenzend an das Aneurysma (Borderzone) und im Myokard weitab von dem Aneurysma wurden durch Minimierung der Fehler zwischen FE Modell vorhergesagt und gemessene systolische Belastungen und LV-Volumes, die mit die Oberfl&auml;che aufeinander folgenden Antwort-Methode zur Optimierung gesch&auml;tzt. Die bedeutende Depression in optimierten T(max_B) relativ T(max_R) wurde durch direkte ex Vivo-Kraft-Messungen von hellh&auml;utigen Faser Vorbereitungen best&auml;tigt. Die optimierten Werte von T(max_B) und T(max_R) waren nicht &uuml;berm&auml;&szlig;ig sensibel auf die passive Materialparameter angegeben. Die Rechenzeit von weniger als 5 Stunden, verbunden mit unserer vorgeschlagenen Methode zur Einsch&auml;tzung der regionalen myokardialen Kontraktilit&auml;t in-vivo macht es klinische potenziell sehr n&uuml;tzlich.

Eine rechnerisch effizienten formale Optimierung der regionalen myokardialen Kontraktilit&auml;t in ein Schaf mit links ventrikul&auml;ren Aneurysma.

Rechtsventrikul&auml;re (RV) Scheitern ist gemeinsame klinische Adjektiv, das schlecht verstanden wird. Damit versagt und Nonfailing Herzen, haben wir untersucht, die unverwechselbaren Inotrope Reaktionen induziert im Myokard RV nach Stimulation von Alpha (1)-adrenerge Rezeptoren (ARs). RV Trabeculae vom Nonfailing Maus Herzen, alpha 1-ARs induziert eine negative Inotrope Antwort, Einklang mit unserer vorherigen Studie. Im Gegensatz, im RV Trabeculae fehlschl&auml;gt, Herzen induzierte 12 Wch nach koronaren Ligaturen, alpha 1-ARs positiv inotrope. Mechanistisch, zeigten Experimente mit hellh&auml;utigen Trabeculae alpha 1-ARs Myofilament Ca(2+) Empfindlichkeit im nonfailing RV Myokard, verringerte, w&auml;hrend alpha 1-ARs Ca(2+) Empfindlichkeit in Herzinsuffizienz erh&ouml;ht. Dies l&auml;sst vermuten, dass ein Wechsel in die Ca(2+) Empfindlichkeit Reaktion auf alpha-1-AR-Stimulation den Schalter in der RV-alpha 1-AR Inotrope Antwort in Herzinsuffizienz erkl&auml;rt. Myosin-Leichtketten-Kinase (MLCK) erh&ouml;hen die Myofilament Ca(2+) Empfindlichkeit und der glatten Muskulatur-Isoform (SmMLCK), das ist auch in Cardiomyocytes, war h&auml;ufiger im RV Myokard von fehlerhaften gegen&uuml;ber nonfailing Herzen. Dar&uuml;ber hinaus verhindert die MLCK Inhibitor ML-9 den Schalter des RV Myokard zu einer positiven alpha 1-AR Inotrope Reaktion bei Herzinsuffizienz. Im linken ventrikul&auml;ren Myokard dagegen alpha 1-AR Inotrope Antworten waren nicht anders in versagt im Vergleich zu nonfailing Herzen und SmMLCK F&uuml;lle war in Herzinsuffizienz nicht erh&ouml;ht. In Bezug auf Krankheiten des Menschen fanden wir, dass SmMLCK-mRNA und Protein-Ebene in RVs verl&auml;ngert wurde, von gescheiterten menschliche Herzen. Wir schlie&szlig;en, dass die RV Inotrope Reaktion auf alpha-1-ARs von negativ zu positiv in Herzinsuffizienz, durch einen Weg mit erh&ouml;hter Myofilament Ca(2+) Empfindlichkeit eingeschaltet ist. Da alpha-1-AR-Agonist Katecholamine Herzinsuffizienz erhoben werden, erh&ouml;hte alpha-1-AR Inotrope Antworten im Myokard RV m&ouml;glicherweise adaptiv Herzinsuffizienz durch Unterst&uuml;tzung bei der fehlerhaften RV reagieren auf pulmonale Druck erh&ouml;ht.

Herzinsuffizienz schaltet die RV alpha1-adrenergene Inotrope Antwort vom negativen ins positive.

Right ventricular (RV) failure is a serious common clinical problem that is poorly understood. Therefore, for failing and nonfailing hearts, we examined the distinctive inotropic responses induced in the RV myocardium after the stimulation of alpha(1)-adrenergic receptors (ARs). In RV trabeculae from nonfailing mouse hearts, alpha(1)-ARs induced a negative inotropic response, consistent with our previous study. In marked contrast, in RV trabeculae from failing hearts, 12 wk after coronary artery ligation, alpha(1)-ARs induced a positive inotropic response. Mechanistically, experiments with skinned trabeculae showed that alpha(1)-ARs decreased myofilament Ca(2+) sensitivity in the nonfailing RV myocardium, whereas alpha(1)-ARs increased Ca(2+) sensitivity in heart failure. This suggests that a switch in the Ca(2+) sensitivity response to alpha(1)-AR stimulation explained the switch in the RV alpha(1)-AR inotropic response in heart failure. Myosin light chain kinase (MLCK) can increase myofilament Ca(2+) sensitivity, and the smooth muscle isoform (smMLCK), which is also present in cardiomyocytes, was more abundant in the RV myocardium from failing versus nonfailing hearts. Moreover, the MLCK inhibitor ML-9 prevented the switch of the RV myocardium to a positive alpha(1)-AR inotropic response in heart failure. In the left ventricular myocardium, in contrast, alpha(1)-AR inotropic responses were not different in failing versus nonfailing hearts, and smMLCK abundance was not increased in heart failure. In relation to human disease, we found that smMLCK mRNA and protein levels were increased in RVs from failing human hearts. We conclude that the RV inotropic response to alpha(1)-ARs is switched from negative to positive in heart failure, through a pathway involving increased myofilament Ca(2+) sensitivity. Since alpha(1)-AR agonist catecholamines are elevated in heart failure, increased alpha(1)-AR inotropic responses in the RV myocardium may be adaptive in heart failure by helping the failing RV respond to increased pulmonary pressures.

Heart failure switches the RV alpha1-adrenergic inotropic response from negative to positive.

Global activation of MAP kinases has been reported in both human and experimental heart failure. Chronic remodeling of the surviving ventricular wall after myocardial infarction (MI) involves both myocyte loss and fibrosis; we hypothesized that this cardiomyopathy involves differential shifts in pro- and anti-apoptotic MAP kinase signaling in cardiac myocyte (CM) and non-myocyte. Cardiomyopathy after coronary artery ligation in mice was characterized by echocardiography, ex vivo Langendorff preparation, histologic analysis and measurements of apoptosis. Phosphorylation (activation) of signaling molecules was analyzed by Western blot, ELISA and immunohistochemistry. Post-MI remodeling involved dramatic changes in the phosphorylation of both stress-activated MAP (SAP) kinase p38 as well as ERK, a known mediator of cell survival, but not of SAP kinase JNK or the anti-apoptotic mediator of PI3K, Akt. Phosphorylation of p38 rose early after MI in the infarct, whereas a more gradual rise in the remote myocardium accompanied a rise in apoptosis in that region. In both areas, ERK phosphorylation was lowest early after MI and rose steadily thereafter, though infarct phosphorylation was consistently higher. Immunostaining of p-ERK localized to fibrotic areas populated primarily by non-myocytes, whereas staining of p38 phosphorylation was stronger in areas of progressive CM apoptosis. Relative segregation of CMs and non-myocytes in different regions of the post-MI myocardium revealed signaling patterns that imply cell type-specific changes in pro- and anti-apoptotic MAP kinase signaling. Prevention of myocyte loss and of LV remodeling after MI may therefore require cell type-specific manipulation of p38 and ERK activation.

Distinctive ERK and p38 signaling in remote and infarcted myocardium during post-MI remodeling in the mouse.

Globale Aktivierung der MAP-Kinasen wurde in sowohl menschlich als auch experimentelle Herzinsuffizienz berichtet. Chronische Umbau des &Uuml;berlebenden Ventrikelwand nach einem Myokardinfarkt (MI) umfasst Muskelfaser Verlust und Fibrose; wir die Hypothese, dass diese Kardiomyopathie differentielle Verschiebungen in der Pro - und Anti-apoptotischen MAP-Kinase signalisieren in kardialen Muskelfaser (CM) und nicht-Muskelfaser beinhaltet. Kardiomyopathie nach koronaren Verbindung in den M&auml;usen war gepr&auml;gt durch Echokardiographie, ex Vivo Langendorff Vorbereitung, histologische Analyse und Messungen von Apoptosis. Phosphorylierung (Aktivierung) signalisieren der Molek&uuml;le wurde von Western-Blot, ELISA und Immunhistochemie analysiert. Post-MI Umbau beteiligte dramatische Ver&auml;nderungen in der Phosphorylierung von Druck aktivierte MAP (SAP) Kinase p38 sowie ERK, bekannten Vermittler der Zelle &uuml;berleben, aber nicht der SAP-Kinase JNK oder die Anti-Apoptotic Vermittler von PI3K, Akt. Phosphorylierung von p38 stieg fr&uuml;h nach MI in der Infarkt, ein allm&auml;hlicher Anstieg der entfernten Myokard einen Anstieg der Apoptose in dieser Region begleitet. In beiden Bereichen ERK Phosphorylierung war fr&uuml;h nach MI am niedrigsten und stieg kontinuierlich danach, obwohl Infarkt Phosphorylierung konstant h&ouml;her war. Immunf&auml;rbung von p-ERK auf fibrotische in erster Linie von nicht-Muskelzellen, bev&ouml;lkerten Gebieten lokalisiert, w&auml;hrend Anbluten von p38 Phosphorylierung in Bereichen von progressive CM Apoptosis st&auml;rker war. Relative Trennung von CMs und nicht-Muskelzellen in verschiedenen Regionen der Post-MI Myokard offenbart Signalling Muster, die die Zelle Typ-spezifische &Auml;nderungen in Pro - und Anti-apoptotischen MAP Kinase signalisieren implizieren. Vermeidung von Myocyte Verlust und der LV Umgestaltung nach MI kann daher Zelle typspezifische Manipulation der p38 und ERK Aktivierung erfordern.

Article	Year
Alpha(1)-adrenoceptor subtypes mediate negative inotropy in myocardium from alpha(1A/C)-knockout and wild type mice. Journal of molecular and cellular cardiology\| PubMed ID: 12234770	2002
Alpha 1-adrenergic receptor responses in alpha 1AB-AR knockout mouse hearts suggest the presence of alpha 1D-AR. American journal of physiology. Heart and circulatory physiology\| PubMed ID: 12595294	2003
Abnormal myocardial contraction in alpha(1A)- and alpha(1B)-adrenoceptor double-knockout mice. Journal of molecular and cellular cardiology\| PubMed ID: 14519431	2003
Cardiac transgenesis with the tetracycline transactivator changes myocardial function and gene expression. Physiological genomics\| PubMed ID: 15797971	2005
Cardiac transgenic matrix metalloproteinase-2 expression directly induces impaired contractility. Cardiovascular research\| PubMed ID: 16183043	2006
Sustained preconditioning induced by cardiac transgenesis with the tetracycline transactivator. American journal of physiology. Heart and circulatory physiology\| PubMed ID: 16243914	2006
Alpha1-adrenergic receptors prevent a maladaptive cardiac response to pressure overload. The Journal of clinical investigation\| PubMed ID: 16585965	2006
Contrasting inotropic responses to alpha1-adrenergic receptor stimulation in left versus right ventricular myocardium. American journal of physiology. Heart and circulatory physiology\| PubMed ID: 16731650	2006
Transgenic MMP-2 expression induces latent cardiac mitochondrial dysfunction. Biochemical and biophysical research communications\| PubMed ID: 17475219	2007
Expression of a Gi-coupled receptor in the heart causes impaired Ca2+ handling, myofilament injury, and dilated cardiomyopathy. American journal of physiology. Heart and circulatory physiology\| PubMed ID: 17965283	2008
A computationally efficient formal optimization of regional myocardial contractility in a sheep with left ventricular aneurysm. Journal of biomechanical engineering\| PubMed ID: 20016753	2009
Heart failure switches the RV alpha1-adrenergic inotropic response from negative to positive. American journal of physiology. Heart and circulatory physiology\| PubMed ID: 20035030	2010
Distinctive ERK and p38 signaling in remote and infarcted myocardium during post-MI remodeling in the mouse. Journal of cellular biochemistry\| PubMed ID: 20186881	2010
Refueling the heart: Using 2-deoxy-ATP to enhance cardiac contractility. Journal of molecular and cellular cardiology\| PubMed ID: 22001677	2011

Anthony J. Baker

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