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

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.

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

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.

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

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.

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

OBJECTIVE: 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. METHODS: Morphologic and functional studies were carried out using both intact and demembranated (skinned) right ventricular trabeculae dissected from hearts of 8-month-old MMP-2 transgenic mice and wild-type controls (WT). RESULTS: Electron micrographs showed that compared to WT, MMP-2 myocardium had no gross, ultrastructural changes (no myocyte dropout or gross fibrosis). However, MMP-2 myocardium contained fibroblasts with abundant rough endoplasmic reticulum, consistent with an activated synthetic phenotype, suggesting extracellular matrix remodeling in MMP-2 trabeculae. Consistent with remodeling, mechanical studies found increased stiffness of intact unstimulated trabeculae (increasing sarcomere lengths from 2 to 2.3 microm caused a greater rise of passive muscle force for MMP-2 trabeculae versus WT). With electrical stimulation, MMP-2 trabeculae generated substantially less active force at all sarcomere lengths. Moreover, inotropic responses to increases of bath [Ca2+], pacing frequency, and isoproterenol were all significantly reduced versus WT trabeculae. Skinned fiber assessment of myofilament function revealed that maximum Ca2+-activated force of skinned MMP-2 trabeculae was reduced to approximately 50% of WT, suggesting a myofilament contraction defect. CONCLUSION: Cardiac-specific, constitutively active MMP-2 expression leads to impaired contraction and diminished responses to inotropic stimulation. These findings indicate that MMP-2 can directly impair ventricular function in the absence of superimposed injury.

Sustained preconditioning induced by cardiac transgenesis with the tetracycline transactivator.

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.

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

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.

Differential effects of stable prostacyclin analogs on smooth muscle proliferation and cyclic AMP generation in human pulmonary artery.

Primary pulmonary hypertension is characterized by increased pulmonary vascular resistance and smooth muscle proliferation. Stable analogs are increasingly being used to treat this disease, although no data exists comparing their effects on proliferation. We therefore investigated the antiproliferative activity of several prostacyclin (PGI(2)) analogs on human pulmonary arterial smooth muscle cells, including UT-15 and iloprost, analogs that have recently completed successful clinical trials. Serum-induced proliferation, as assessed by [(3)H]thymidine incorporation (30 h) or cell number (48 h), was significantly inhibited with a 10-fold difference in potency, ranking in effectiveness UT-15 > iloprost > cicaprost > beraprost. Effects were reversed by the adenylyl cyclase inhibitor, 2,5'dideoxyadenosine (DDA) but not SQ22536. Intracellular cyclic AMP (cAMP) was elevated by all analogs and inhibited by DDA, although SQ22536 was a highly variable inhibitor, suggesting that different pathways might mediate cAMP generation. UT-15 produced a significantly larger and more sustained increase in cAMP compared with other analogs, with iloprost being the weakest elevator. Thus, PGI(2) analogs potently inhibit proliferation of human pulmonary artery, probably via a cAMP-dependent pathway, although cAMP elevation in itself is not a good predictor of antiproliferative potency.