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In JoVE (1)
Other Publications (12)
- Journal of Molecular and Cellular Cardiology
- Nature
- American Journal of Physiology. Heart and Circulatory Physiology
- Journal of Molecular and Cellular Cardiology
- Physiological Genomics
- Cardiovascular Research
- American Journal of Physiology. Heart and Circulatory Physiology
- The Journal of Clinical Investigation
- American Journal of Physiology. Heart and Circulatory Physiology
- American Journal of Physiology. Heart and Circulatory Physiology
- Journal of Cellular Biochemistry
- Magnetic Resonance in Medicine : Official Journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine
Articles by Paul C. Simpson in JoVE
JoVE General
Synthesis of an In vivo MRI-detectable Apoptosis Probe
1Division of Cardiovascular Medicine, Department of Medicine, Stanford University Medical Center, 2Division of Cardiology, Department of Medicine, University of California, San Francisco, 3San Francisco VAMC
Early detection of apoptosis may identify at-risk cell populations in a variety of diseases. Here we demonstrate a method to link an early apoptosis-detection protein (Annexin V) to a MRI-detectable iron oxide nanoparticle (SPIO). This method may be extended to other proteins of interest to generate MRI-detectable molecular imaging probes.
Other articles by Paul C. Simpson on PubMed
Alpha(1)-adrenoceptor Subtypes Mediate Negative Inotropy in Myocardium from Alpha(1A/C)-knockout and Wild Type Mice
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.
Overview of the Alliance for Cellular Signaling
The Alliance for Cellular Signaling is a large-scale collaboration designed to answer global questions about signalling networks. Pathways will be studied intensively in two cells--B lymphocytes (the cells of the immune system) and cardiac myocytes--to facilitate quantitative modelling. One goal is to catalyse complementary research in individual laboratories; to facilitate this, all alliance data are freely available for use by the entire research community.
Alpha 1-adrenergic Receptor Responses in Alpha 1AB-AR Knockout Mouse Hearts Suggest the Presence of Alpha 1D-AR
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.
Abnormal Myocardial Contraction in Alpha(1A)- and Alpha(1B)-adrenoceptor Double-knockout Mice
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.
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.
Cardiac Transgenic Matrix Metalloproteinase-2 Expression Directly Induces Impaired Contractility
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.
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.
Alpha1-adrenergic Receptors Prevent a Maladaptive Cardiac Response to Pressure Overload
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.
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.
Heart Failure Switches the RV Alpha1-adrenergic Inotropic Response from Negative to 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.
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.
A Molecular MRI Probe to Detect Treatment of Cardiac Apoptosis in Vivo
Cell death by apoptosis is critical in myocardial diseases, and noninvasive detection of early, reversible apoptosis might be useful clinically. Exogenous Annexin-V (ANX) protein binds membrane phosphatidylserine, which is externalized in early apoptosis. A molecular MRI probe was constructed with superparamagnetic iron oxide (SPIO) conjugated to recombinant human ANX (ANX-SPIO). Apoptosis was induced with doxorubicin, a cardiotoxic cancer drug, in culture in neonatal rat ventricular myocytes, cardiac fibroblasts, and mesenchymal stem cells, and in vivo in the mouse heart. ANX-SPIO was validated using T2*-weighted 3T MRI. ANX-SPIO produced T2* signal loss, reflecting iron content, that correlated highly with independent apoptosis markers; bound with high affinity to apoptotic myocytes by competition assay (Ki 69 nM); detected apoptosis in culture much earlier than did TUNEL stain; and revealed fibroblast resistance to apoptosis. With apoptosis in vivo, ANX-SPIO produced diffuse myocardial T2* signal loss that correlated with increased iron stain and caspase activity. Treatment with an alpha-1-adrenergic agonist in vivo reversed apoptosis and eliminated the ANX-SPIO MRI signal. It is concluded that cardiac MRI of ANX-SPIO detects early, nonischemic cardiac apoptosis in culture and in vivo, and can identify reversibly injured cardiac cells in diseased hearts, when treatment is still possible.
