Reduced cardiac muscle power with low ATP simulating heart failure.

For patients with heart failure, myocardial ATP level can be reduced to one-half of that observed in healthy controls. This marked reduction (from ≈8 mM in healthy controls to as low as 3-4 mM in heart failure) has been suggested to contribute to impaired myocardial contraction and to the decreased pump function characteristic of heart failure. However, in vitro measures of maximum myofilament force generation, maximum shortening velocity, and the actomyosin ATPase activity show effective KM values for MgATP ranging from ≈10 µM to 150 µM, well below the intracellular ATP level in heart failure. Thus, it is not clear that the fall of myocardial ATP observed in heart failure is sufficient to impair the function of the contractile proteins. Therefore, we tested the effect of low MgATP levels on myocardial contraction using demembranated cardiac muscle preparations that were exposed to MgATP levels typical of the range found in non-failing and failing hearts. Consistent with previous studies, we found that a 50% reduction in MgATP level (from 8 mM to 4 mM) did not reduce maximum force generation or maximum velocity of shortening. However, we found that a 50% reduction in MgATP level caused a 20%-25% reduction in maximal power generation (measured during muscle shortening against a load) and a 20% slowing of cross-bridge cycling kinetics. These results suggest that the decreased cellular ATP level occurring in heart failure contributes to the impaired pump function of the failing heart. Since the ATP-myosin ATPase dissociation constant is estimated to be submillimolar, these findings also suggest that MgATP concentration affects cross-bridge dynamics through a mechanism that is more complex than through the direct dependence of MgATP concentration on myosin ATPase activity. Finally, these studies suggest that therapies targeted to increase adenine nucleotide pool levels in cardiomyocytes might be beneficial for treating heart failure.

Coupling to Gq Signaling Is Required for Cardioprotection by an Alpha-1A-Adrenergic Receptor Agonist.

RATIONALE: Gq signaling in cardiac myocytes is classically considered toxic. Targeting Gq directly to test this is problematic, because cardiac myocytes have many Gq-coupled receptors. OBJECTIVE: Test whether Gq coupling is required for the cardioprotective effects of an alpha-1A-AR (adrenergic receptor) agonist. METHODS AND RESULTS: In recombinant cells, a mouse alpha-1A-AR with a 6-residue substitution in the third intracellular loop does not couple to Gq signaling. Here we studied a knockin mouse with this alpha-1A-AR mutation. Heart alpha-1A receptor levels and antagonist affinity in the knockin were identical to wild-type. In wild-type cardiac myocytes, the selective alpha-1A agonist A61603-stimulated phosphoinositide-phospholipase C and myocyte contraction. In myocytes with the alpha-1A knockin, both A61603 effects were absent, indicating that Gq coupling was absent. Surprisingly, A61603 activation of cardioprotective ERK (extracellular signal-regulated kinase) was markedly impaired in the KI mutant myocytes, and A61603 did not protect mutant myocytes from doxorubicin toxicity in vitro. Similarly, mice with the α1A KI mutation had increased mortality after transverse aortic constriction, and A61603 did not rescue cardiac function in mice with the Gq coupling-defective alpha-1A receptor. CONCLUSIONS: Gq coupling is required for cardioprotection by an alpha-1A-AR agonist. Gq signaling can be adaptive.

Monitoring the Health Status of Mice with Bleomycin-induced Lung Injury by Using Body Condition Scoring.

Well-defined, humane endpoints aid in monitoring animal health status during disease development. Body condition scoring (BCS) is a method for assessing health status in mouse studies where wasting and death are potential endpoints. Whether BCS is useful in monitoring animals with bleomycin-induced lung injury has not been reported. Body weight (BW) is a common humane endpoint for this model, but because the lungs increase in weight as BW decreases, the animal's true physical condition could be masked when using BW as the sole endpoint criterion. Therefore, our goal here was to assess the usefulness of BCS in monitoring health status in a mouse model of lung injury. Lung injury was caused by acute instil- lation of the fibrogenic antibiotic bleomycin into the lungs through the trachea. Male C57BL/6 mice received bleomycin (0.075 U) dissolved in saline or saline alone. Bleomycin instillation led to a doubling of lung weight and decreases in both BW and BCS, compared with saline instillation. The changes in BW and BCS were significantly correlated with lung weight. When the adjusted BW was used (corrected for the increase in lung weight), the correlation was unchanged, suggesting that the increase in lung weight did not significantly mask the decrease in BW. Bleomycin instillation caused decreases in both soleus and visceral epididymal fat masses. The change in BCS was significantly correlated with both soleus and VEF mass, suggesting that BCS is reflective of the systemic loss of muscle and fat mass. Our findings suggest that BW and BCS are significantly correlated to lung injury in the bleomycin model of lung fibrosis and that BCS is an appropriate alternative humane endpoint in this mouse model.

Reversal of right ventricular failure by chronic α1A-subtype adrenergic agonist therapy.

Right ventricular (RV) failure (RVF) is a serious disease with no effective treatment available. We recently reported a disease prevention study showing that chronic stimulation of α1A-adrenergic receptors (α1A-ARs), started at the time of RV injury, prevented the development of RVF. The present study used a clinically relevant disease reversal design to test if chronic α1A-AR stimulation, started after RVF was established, could reverse RVF. RVF was induced surgically by pulmonary artery constriction in mice. Two weeks after pulmonary artery constriction, in vivo RV fractional shortening as assessed by MRI was reduced by half relative to sham-operated controls (25 ± 2%, n = 27, vs. 52 ± 2%, n = 13, P < 10-11). Subsequent chronic treatment with the α1A-AR agonist A61603 for a further 2 wk resulted in a substantial recovery of RV fractional shortening (to 41 ± 2%, n = 17, P < 10-7 by a paired t-test) along with recovery of voluntary exercise capacity. Mechanistically, chronic A61603 treatment resulted in increased activation of the prosurvival kinase ERK, increased abundance of the antiapoptosis factor Bcl-2, and decreased myocyte necrosis evidenced by a decreased serum level of cardiac troponin. Moreover, A61603 treatment caused increased abundance of the antioxidant glutathione peroxidase-1, decreased level of reactive oxygen species, and decreased oxidative modification (carbonylation) of myofilament proteins. Consistent with these effects, A61603 treatment resulted in increased force development by cardiac myofilaments, which might have contributed to increased RV function. These findings suggest that the α1A-AR is a therapeutic target to reverse established RVF. NEW & NOTEWORTHY Currently, there are no effective therapies for right ventricular (RV) failure (RVF). This project evaluated a novel therapy for RVF. In a mouse model of RVF, chronic stimulation of α1A-adrenergic receptors with the agonist A61603 resulted in recovery of in vivo RV function, improved exercise capacity, reduced oxidative stress-related carbonylation of contractile proteins, and increased myofilament force generation. These results suggest that the α1A-adrenergic receptor is a therapeutic target to treat RVF.

A Myocardial Slice Culture Model Reveals Alpha-1A-Adrenergic Receptor Signaling in the Human Heart.

BACKGROUND: Translation of preclinical findings could benefit from a simple, reproducible, high throughput human model to study myocardial signaling. Alpha-1A-adrenergic receptors (ARs) are expressed at very low levels in the human heart, and it is unknown if they function. OBJECTIVES: To develop a high throughput human myocardial slice culture model, and to test the hypothesis that alpha-1A- ARs are functional in the human heart. METHODS: Cores of LV free wall 8 mm diameter were taken from 52 hearts (18 failing and 34 nonfailing). Slices 250 µm thick were cut with a Krumdieck apparatus and cultured using a rotating incubation unit. RESULTS: About 60 slices were cut from each LV core, and a typical study could use 96 slices. Myocyte morphology was maintained, and diffusion into the slice center was rapid. Slice viability was stable for at least 3 days in culture by ATP and MTT assays. The beta-AR agonist isoproterenol stimulated phospholamban phosphorylation, and the alpha-1A-AR agonist A61603 stimulated ERK phosphorylation, with nanomolar EC50 values in slices from both failing and nonfailing hearts. Strips cut from the slices were used to quantify activation of contraction by isoproterenol, A61603, and phenylephrine. The slices supported transduction by adenovirus. CONCLUSIONS: We have developed a simple, high throughput LV myocardial slice culture model to study signaling in the human heart. This model can be useful for translational studies, and we show for the first time that the alpha-1A-AR is functional in signaling and contraction in the human heart.

The α1A-adrenergic receptor subtype mediates increased contraction of failing right ventricular myocardium.

Dysfunction of the right ventricle (RV) is closely related to prognosis for patients with RV failure. Therefore, strategies to improve failing RV function are significant. In a mouse RV failure model, we previously reported that α1-adrenergic receptor (α1-AR) inotropic responses are increased. The present study determined the roles of both predominant cardiac α1-AR subtypes (α1A and α1B) in upregulated inotropy in failing RV. We used the mouse model of bleomycin-induced pulmonary fibrosis, pulmonary hypertension, and RV failure. We assessed the myocardial contractile response in vitro to stimulation of the α1A-subtype (using α1A-subtype-selective agonist A61603) and α1B-subtype [using α1A-subtype knockout mice and nonsubtype selective α1-AR agonist phenylephrine (PE)]. In wild-type nonfailing RV, a negative inotropic effect of α1-AR stimulation with PE (force decreased ≈50%) was switched to a positive inotropic effect (PIE) with bleomycin-induced RV injury. Upregulated inotropy in failing RV occurred with α1A-subtype stimulation (force increased ≈200%), but not with α1B-subtype stimulation (force decreased ≈50%). Upregulated inotropy mediated by the α1A-subtype involved increased activator Ca(2+) transients and increased phosphorylation of myosin regulatory light chain (a mediator of increased myofilament Ca(2+) sensitivity). In failing RV, the PIE elicited by the α1A-subtype was appreciably less when the α1A-subtype was stimulated in combination with the α1B-subtype, suggesting functional antagonism between α1A- and α1B-subtypes. In conclusion, upregulation of α1-AR inotropy in failing RV myocardium requires the α1A-subtype and is opposed by the α1B-subtype. The α1A subtype might be a therapeutic target to improve the function of the failing RV.

Myofilament dysfunction contributes to impaired myocardial contraction in the infarct border zone.

After myocardial infarction, a poorly contracting nonischemic border zone forms adjacent to the infarct. The cause of border zone dysfunction is unclear. The goal of this study was to determine the myofilament mechanisms involved in postinfarction border zone dysfunction. Two weeks after anteroapical infarction of sheep hearts, we studied in vitro isometric and isotonic contractions of demembranated myocardium from the infarct border zone and a zone remote from the infarct. Maximal force development (Fmax) of the border zone myocardium was reduced by 31 ± 2% versus the remote zone myocardium (n = 6/group, P < 0.0001). Decreased border zone Fmax was not due to a reduced content of contractile material, as assessed histologically, and from myosin content. Furthermore, decreased border zone Fmax did not involve altered cross-bridge kinetics, as assessed by muscle shortening velocity and force development kinetics. Decreased border zone Fmax was associated with decreased cross-bridge formation, as assessed from muscle stiffness in the absence of ATP where cross-bridge formation should be maximized (rigor stiffness was reduced 34 ± 6%, n = 5, P = 0.011 vs. the remote zone). Furthermore, the border zone myocardium had significantly reduced phosphorylation of myosin essential light chain (ELC; 41 ± 10%, n = 4, P < 0.05). However, for animals treated with doxycycline, an inhibitor of matrix metalloproteinases, rigor stiffness and ELC phosphorylation were not reduced in the border zone myocardium, suggesting that doxycycline had a protective effect. In conclusion, myofilament dysfunction contributes to postinfarction border zone dysfunction, myofilament dysfunction involves impaired cross-bridge formation and decreased ELC phosphorylation, and matrix metalloproteinase inhibition may be beneficial for limiting postinfarct border zone dysfunction.

N-terminal truncated intracellular matrix metalloproteinase-2 induces cardiomyocyte hypertrophy, inflammation and systolic heart failure.

Matrix metalloproteinase-2 (MMP-2) is increasingly recognized as a major contributor to progressive cardiac injury within the setting of ischemia-reperfusion injury and ischemic ventricular remodeling. A common feature of these conditions is an increase in oxidative stress, a process that engages multiple pro-inflammatory and innate immunity cascades. We recently reported on the identification and characterization of an intracellular isoform of MMP-2 generated by oxidative stress-mediated activation of an alternative promoter located within the first intron of the MMP-2 gene. Transcription from this site generates an N-terminal truncated 65 kDa isoform of MMP-2 (NTT-MMP-2) that lacks the secretory sequence and the inhibitory prodomain region. The NTT-MMP-2 isoform is intracellular, enzymatically active and localizes in part to mitochondria. Expression of the NTT-MMP-2 isoform triggers Nuclear Factor of Activated T-cell (NFAT) and NF-κB signaling with the expression of a highly defined innate immunity transcriptome, including Interleukin-6, MCP-1, IRF-7 and pro-apoptotic transcripts. To determine the functional significance of the NTT-MMP-2 isoform in vivo we generated cardiac-specific NTT-MMP-2 transgenic mice. These mice developed progressive cardiomyocyte and ventricular hypertrophy associated with systolic heart failure. Further, there was evidence for cardiomyocyte apoptosis and myocardial infiltration with mononuclear cells. The NTT-MMP-2 transgenic hearts also demonstrated more severe injury following ex vivo ischemia-reperfusion injury. We conclude that a novel intracellular MMP-2 isoform induced by oxidant stress directly contributes, in the absence of superimposed injury, to cardiomyocyte hypertrophy. inflammation, systolic heart failure and enhanced susceptibility to ischemia-reperfusion injury.

Left ventricular myocardial contractility is depressed in the borderzone after posterolateral myocardial infarction.

BACKGROUND: Contractility in the borderzone (BZ) after anteroapical myocardial infarction (MI) is depressed. We tested the hypothesis that BZ contractility is also decreased after posterolateral MI. METHODS: Five sheep underwent posterolateral MI. Magnetic resonance imaging (MRI) was performed 2 weeks before and 16 weeks after MI, and left ventricular (LV) volume and regional strain were measured. Finite element (FE) models were constructed, and the systolic material parameter, Tmax, was calculated in the BZ and remote myocardium by minimizing the difference between experimentally measured and calculated LV strain and volume. Sheep were sacrificed 17 weeks after MI, and myocardial muscle fibers were taken from the BZ and remote myocardium. Fibers were chemically demembranated, and isometric developed force, Fmax, was measured at supramaximal [Ca(2+)]. Routine light microscopy was also performed. RESULTS: There was no difference in Tmax in the remote myocardium before and 16 weeks after MI. However, there was a large decrease (63.3%, p = 0.005) in Tmax in the BZ when compared with the remote myocardium 16 weeks after MI. In addition, there was a significant reduction of BZ Fmax for all samples (18.9%, p = 0.0067). Myocyte cross-sectional area increased by 61% (p = 0.021) in the BZ, but there was no increase in fibrosis. CONCLUSIONS: Contractility in the BZ is significantly depressed relative to the remote myocardium after posterolateral MI. The reduction in contractility is due at least in part to a decrease in contractile protein function.

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.

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.