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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 K

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.

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.

The heart has two intrinsic mechanisms to enhance contractile strength that compensate for increased mechanical load to help maintain cardiac output. When vascular resistance increases the ventricular chamber initially expands causing an immediate length-dependent increase of contraction force via the Frank-Starling mechanism. Additionally, the stress-dependent Anrep effect slowly increases contraction force that results in the recovery of the chamber volume towards its initial state. The Anrep effect poses a paradox: how can the cardiomyocyte maintain higher contractility even after the cell length has recovered its initial length? Here we propose a surface mechanosensor model that enables the cardiomyocyte to sense different mechanical stresses at the same mechanical strain. The cell-surface mechanosensor is coupled to a mechano-chemo-transduction feedback mechanism involving three elements: surface mechanosensor strain, intracellular Ca2+ transient, and cell strain. We show that in this simple yet general system, contractility autoregulation naturally emerges, enabling the cardiomyocyte to maintain contraction amplitude despite changes in a range of afterloads. These nontrivial model predictions have been experimentally confirmed. Hence, this model provides a new conceptual framework for understanding the contractility autoregulation in cardiomyocytes, which contributes to the heart’s intrinsic adaptivity to mechanical load changes in health and diseases.

The decrease in contractility in myocardium adjacent (border zone; BZ) to a myocardial infarction (MI) is correlated with an increase in reactive oxygen species (ROS). We hypothesized that injection of a thermoresponsive hydrogel, with ROS scavenging properties, into the MI would decrease ROS and improve BZ function. Fourteen sheep underwent antero-apical MI. Seven sheep had a comb-like copolymer synthesized from N-isopropyl acrylamide (NIPAAm) and 1500 MW methoxy poly(ethylene glycol) methacrylate, (NIPAAm-PEG1500), injected (20 × 0.5 mL) into the MI zone 40 min after MI (MI + NIPAAm-PEG1500) and seven sheep were MI controls. Cardiac MRI was performed 2 weeks before and 6 weeks after MI + NIPAAm-PEG1500. BZ wall thickness at end systole was significantly higher for MI + NIPAAm-PEG1500 (12.32 ± 0.51 mm/m2 MI + NIPAAm-PEG1500 vs. 9.88 ± 0.30 MI; p = .023). Demembranated muscle force development for BZ myocardium 6 weeks after MI was significantly higher for MI + NIPAAm-PEG1500 (67.67 ± 2.61 mN/m2 MI + NIPAAm-PEG1500 vs. 40.53 ± 1.04 MI; p < .0001) but not significantly different from remote myocardium or BZ or non-operated controls. Levels of ROS in BZ tissue were significantly lower in the MI + NIPAAm-PEG1500 treatment group (hydroxyl p = .0031; superoxide p = .0182). We conclude that infarct injection of the NIPAAm-PEG1500 hydrogel with ROS scavenging properties decreased ROS and improved contractile protein function in the border zone 6 weeks after MI.

Failure of the right ventricle (RV) is a serious disease with a poor prognosis and limited treatment options. Signaling by α1-adrenergic receptors (α1-ARs), in particular the α1A-subtype, mediate cardioprotective effects in multiple heart failure models. Recent studies have shown that chronic treatment with the α1A-subtype agonist A61603 improves function and survival in a model of left ventricular failure. The goal of the present study was to determine if chronic A61603 treatment is beneficial in a RV failure model. We used tracheal instillation of the fibrogenic antibiotic bleomycin in mice to induce pulmonary fibrosis, pulmonary hypertension, and RV failure within 2 wk. Some mice were chronically treated with a low dose of A61603 (10 ng·kg−1·day−1). In the bleomycin model of RV failure, chronic A61603 treatment was associated with improved RV fractional shortening and greater in vitro force development by RV muscle preparations. Cell injury markers were reduced with A61603 treatment (serum cardiac troponin I, RV fibrosis, and expression of matrix metalloproteinase-2). RV oxidative stress was reduced (using the probes dihydroethidium and 4-hydroxynonenal). Consistent with lowered RV oxidative stress, A61603 was associated with an increased level of the cellular antioxidant superoxide dismutase 1 and a lower level of the prooxidant NAD(P)H oxidase isoform NOX4. In summary, in the bleomycin model of RV failure, chronic A61603 treatment reduced RV oxidative stress, RV myocyte necrosis, and RV fibrosis and increased both RV function and in vitro force development. These findings suggest that in the context of pulmonary fibrosis, the α1A-subtype is a potential therapeutic target to treat the failing RV. NEW & NOTEWORTHY Right ventricular (RV) failure is a serious disease with a poor prognosis and no effective treatments. In the mouse bleomycin model of RV failure, we tested the efficacy of a treatment using the α1A-adrenergic receptor subtype agonist A61603. Chronic A61603 treatment improved RV contraction and reduced multiple indexes of RV injury, suggesting that the α1A-subtype is a therapeutic target to treat RV failure.

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 α(1A)-adrenergic receptors (α(1A)-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 α(1A)-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 α(1A)-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 α(1A)-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 α(1A)-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 α(1A)-adrenergic receptor is a therapeutic target to treat RVF.

Recent studies report that a single subtype of α1-adrenergic receptor (α1-AR), the α1A-subtype, mediates robust cardioprotective effects in multiple experimental models of heart failure, suggesting that the α1A-subtype is a potential therapeutic target for an agonist to treat heart failure. Moreover, we recently found that the α1A-subtype is present in human heart. The goal of this study was to assess the inotropic response mediated by the α1A-subtype in human myocardium, and to determine whether the response is downregulated in myocardium from failing human heart. We measured in vitro contractile responses of cardiac muscle preparations (trabeculae) isolated from the right ventricle from nonfailing and failing human hearts. Addition of the α1A-subtype agonist A61603 (100 nM) resulted in a large positive inotropic response (force increased ≈ 2-fold). This response represented ≈70% of the response mediated by the β-adrenergic receptor agonist isoproterenol (1 μM). Moreover, in myocardium from failing hearts, α1A-subtype responses remained robust, and only slightly reduced relative to nonfailing hearts. We conclude that α1A-subtype-mediated inotropy could represent a significant source of inotropic support in the human heart. Furthermore, the α1A-subtype remains functional in myocardium from failing human hearts and thus, might be a therapeutic target to support cardioprotective effects in patients with heart failure.

Right ventricular (RV) failure, which occurs in the setting of pressure overload, is characterized by abnormalities in mechanical and energetic function. The effects of these cell- and tissue-level changes on organ-level RV function are unknown. The primary aim of this study was to investigate the effects of myofiber mechanics and mitochondrial energetics on organ-level RV function in the context of pressure overload using a multiscale model of the cardiovascular system. The model integrates the mitochondria-generated metabolite concentrations that drive intracellular actin-myosin cross-bridging and extracellular myocardial tissue mechanics in a biventricular heart model coupled with simple lumped parameter circulations. Three types of pressure overload were simulated and compared to experimental results. The computational model was able to capture a wide range of cardiovascular physiology and pathophysiology from mild RV dysfunction to RV failure. Our results confirm that, in response to pressure overload alone, the RV is able to maintain cardiac output (CO) and predict that alterations in either RV active myofiber mechanics or RV metabolite concentrations are necessary to decrease CO.

Decreased contractility in the non-ischemic border zone surrounding a MI is in part due to degradation of cardiomyocyte sarcomeric components by intracellular matrix metalloproteinase-2 (MMP-2). We recently reported that MMP-2 levels were increased in the border zone after a MI and that treatment with doxycycline for two weeks after MI was associated with normalization of MMP-2 levels and improvement in ex-vivo contractile protein developed force in the myocardial border zone. The purpose of the current study was to determine if there is a sustained effect of short term treatment with doxycycline (Dox) on border zone function in a large animal model of antero-apical myocardial infarction (MI). Antero-apical MI was created in 14 sheep. Seven sheep received doxycycline 0.8 mg/kg/hr IV for two weeks. Cardiac MRI was performed two weeks before, and then two and six weeks after MI. Two sheep died prior to MRI at six weeks from surgical/anesthesia-related causes. The remaining 12 sheep completed the protocol. Doxycycline induced a sustained reduction in intracellular MMP-2 by Western blot (3649±643 MI+Dox vs 9236±114 MI relative intensity; p = 0.0009), an improvement in ex-vivo contractility (65.3±2.0 MI+Dox vs 39.7±0.8 MI mN/mm2; p

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 Ca2+transients and increased phosphorylation of myosin regulatory light chain (a mediator of increased myofilament Ca2+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.

Recently, we used several assays of expression and function to quantify adrenergic receptors (ARs) in isolated, individual adult mouse ventricular myocytes. We found that the β1 and α1B were present on all cells. The α1A was expressed and functional in a 60% subset, with 20% having high receptor levels, and the β2 and β3 were mostly absent on myocytes, but abundant on nonmyocytes.1 Balligand and Michel raise concerns that we could have missed β3-ARs on myocytes with our assays, instead proposing an important role for the β3-AR in cardiac myocytes (in the letter by Balligand and Michel). We thank our colleagues for calling attention to our article. However, we disagree with their concerns. First, Balligand and Michel note that our mRNA arrays and RT-qPCR might not have detected the C-terminal b isoform of the mouse β3-AR.2 On the contrary, the β3-mRNA probes in our arrays, which detected only 4 of 50 cells positive for the β3 (Figure 6 in the study by Myagmar et al1), targeted the exon common to both the a and b isoforms. Our RT-qPCR, which used intron-spanning primers, focused on the a isoform because the β3 b isoform is an extremely tiny fraction of a low total β3-mRNA in mouse heart,2 and its presence or absence would not alter the conclusions. Our RT-qPCR found no β3 in ventricular myocytes but abundant β3 in nonmyocytes, including endothelial cells (Online Figure V in the study by Myagmar et al1). Second, Balligand and Michel raise concerns that our radioligand …

Right ventricular (RV) failure is a serious disease with a poor prognosis and no effective treatments. In the mouse bleomycin model of RV failure, we tested the efficacy of a treatment using the α1A-adrenergic receptor subtype agonist A61603. Chronic A61603 treatment improved RV contraction and reduced multiple indexes of RV injury, suggesting that the α1A-subtype is a therapeutic target to treat RV failure.

Rationale: It is unknown whether every ventricular myocyte expresses all 5 of the cardiac adrenergic receptors (ARs), β1, β2, β3, α1A, and α1B. The β1 and β2 are thought to be the dominant myocyte ARs. Objective: Quantify the 5 cardiac ARs in individual ventricular myocytes. Methods and Results: We studied ventricular myocytes from wild-type mice, mice with α1A and α1B knockin reporters, and β1 and β2 knockout mice. Using individual isolated cells, we measured knockin reporters, mRNAs, signaling (phosphorylation of extracellular signal–regulated kinase and phospholamban), and contraction. We found that the β1 and α1B were present in all myocytes. The α1A was present in 60%, with high levels in 20%. The β2 and β3 were detected in only ≈5% of myocytes, mostly in different cells. In intact heart, 30% of total β-ARs were β2 and 20% were β3, both mainly in nonmyocytes. Conclusion: The dominant ventricular myocyte ARs present in all cells are the β1 and α1B. The β2 and β3 are mostly absent in myocytes but are abundant in nonmyocytes. The α1A is in just over half of cells, but only 20% have high levels. Four distinct myocyte AR phenotypes are defined: 30% of cells with β1 and α1B only; 60% that also have the α1A; and 5% each that also have the β2 or β3. The results raise cautions in experimental design, such as receptor overexpression in myocytes that do not express the AR normally. The data suggest new paradigms in cardiac adrenergic signaling mechanisms.

Adrenergic receptors (AR) are G-protein-coupled receptors (GPCRs) that have a crucial role in cardiac physiology in health and disease. Alpha1-ARs signal through Gαq, and signaling through Gq, for example, by endothelin and angiotensin receptors, is thought to be detrimental to the heart. In contrast, cardiac alpha1-ARs mediate important protective and adaptive functions in the heart, although alpha1-ARs are only a minor fraction of total cardiac ARs. Cardiac alpha1-ARs activate pleiotropic downstream signaling to prevent pathologic remodeling in heart failure. Mechanisms defined in animal and cell models include activation of adaptive hypertrophy, prevention of cardiac myocyte death, augmentation of contractility, and induction of ischemic preconditioning. Surprisingly, at the molecular level, alpha1-ARs localize to and signal at the nucleus in cardiac myocytes, and, unlike most GPCRs, activate "inside-out" signaling to cause cardioprotection. Contrary to past opinion, human cardiac alpha1-AR expression is similar to that in the mouse, where alpha1-AR effects are seen most convincingly in knockout models. Human clinical studies show that alpha1-blockade worsens heart failure in hypertension and does not improve outcomes in heart failure, implying a cardioprotective role for human alpha1-ARs. In summary, these findings identify novel functional and mechanistic aspects of cardiac alpha1-AR function and suggest that activation of cardiac alpha1-AR might be a viable therapeutic strategy in heart failure.

Background—Contractility in the borderzone (BZ) after antero-apical myocardial infarction (MI) is depressed. We tested the hypothesis that BZ contractility is also decreased after posterolateral MI. Methods—Five sheep underwent postero-lateral 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 supra-maximal [Ca 2+ ]. Routine light microscopy was also performed. Results—There was no difference in Tmax between pre-MI and remote myocardium 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=0.023). Myocyte cross-sectional area increased by 61% (p=0.021) in the BZ, but there was no increase in fibrosis. Conclusion—Contractility in the borderzone is significantly depressed relative to the remote myocardium after postero-lateral MI. The reduction in contractility is due at least in part to a decrease in contractile protein function.

α1-Adrenergic receptors (α1-ARs) elicit a negative inotropic effect (NIE) in the mouse right ventricular (RV) myocardium but a positive inotropic effect (PIE) in the left ventricular (LV) myocardium. Effects on myofilament Ca2+sensitivity play a role, but effects on Ca2+handling could also contribute. We monitored the effects of α1-AR stimulation on contraction and Ca2+transients using single myocytes isolated from the RV or LV. Interestingly, for both the RV and LV, we found heterogeneous myocyte inotropic responses. α1-ARs mediated either a PIE or NIE, although RV myocytes had a greater proportion of cells manifesting a NIE (68%) compared with LV myocytes (36%). Stimulation of a single α1-AR subtype (α1A-ARs) with a subtype-selective agonist also elicited heterogeneous inotropic responses, suggesting that the heterogeneity arose from events downstream of the α1A-AR subtype. For RV and LV myocytes, an α1-AR-mediated PIE was associated with an increased Ca2+transient and a NIE was associated with a decreased Ca2+transient, suggesting a key role for Ca2+handling. For RV and LV myocytes, α1-AR-mediated decreases in the Ca2+transient were associated with increased Ca2+export from the cell and decreased Ca2+content of the sarcoplasmic reticulum. In contrast, for myocytes with α1-AR-induced increased Ca2+transients, sarcoplasmic reticulum Ca2+content was not increased, suggesting that other mechanisms contributed to the increased Ca2+transients. This study demonstrates the marked heterogeneity of LV and RV cellular inotropic responses to stimulation of α1-ARs and reveals a new aspect of biological heterogeneity among myocytes in the regulation of contraction.

Acute kidney injury (AKI) is a major risk factor for the development of chronic kidney disease (CKD). Persistent oxidative stress and mitochondrial dysfunction are implicated across diverse forms of AKI and in the transition to CKD. In this study, we applied hyperpolarized (HP) 13C dehydroascorbate (DHA) and 13C pyruvate MR spectroscopy to investigate the renal redox capacity and mitochondrial pyruvate dehydrogenase (PDH) activity, respectively, in a murine model of AKI at baseline, and 7 days after unilateral ischemia reperfusion injury (IRI). Compared to the contralateral sham-operated kidneys, the kidneys subjected to IRI showed a significant decrease in the HP 13C Vitamin C/(Vitamin C+DHA) ratio, consistent with a decrease in redox capacity. The kidneys subjected to IRI also showed a significant decrease in the HP 13C bicarbonate/pyruvate ratio, consistent with impaired PDH activity. The IRI kidneys showed a significantly higher HP 13C lactate/pyruvate ratio at day 7 compared to baseline, although the 13C lactate/pyruvate ratio was not significantly different between the IRI and the contralateral sham-operated kidneys at day 7. Arterial spin labeling MRI demonstrated significantly reduced perfusion in the IRI kidneys. Renal tissue analysis showed corresponding increased reactive oxygen species (ROS), and reduced PDH activity in the IRI kidneys. Our results show the feasibility of HP 13C MR for the noninvasive assessment of oxidative stress and mitochondrial PDH activity following renal ischemia reperfusion injury.

Acute kidney injury (AKI) causes severe morbidity, mortality, and chronic kidney disease (CKD). Mortality is particularly marked in the elderly and with preexisting CKD. Oxidative stress is a common theme in models of AKI induced by ischemia-reperfusion (I-R) injury. We recently characterized an intracellular isoform of matrix metalloproteinase-2 (MMP-2) induced by oxidative stress-mediated activation of an alternate promoter in the first intron of the MMP-2 gene. This generates an NH2-terminal truncated MMP-2 (NTT-MMP-2) isoform that is intracellular and associated with mitochondria. The NTT-MMP-2 isoform is expressed in kidneys of 14-mo-old mice and in a mouse model of coronary atherosclerosis and heart failure with CKD. We recently determined that NTT-MMP-2 is induced in human renal transplants with delayed graft function and correlated with tubular cell necrosis. To determine mechanism(s) of action, we generated proximal tubule cell-specific NTT-MMP-2 transgenic mice. Although morphologically normal at the light microscopic level at 4 mo, ultrastructural studies revealed foci of tubular epithelial cell necrosis, the mitochondrial permeability transition, and mitophagy. To determine whether NTT-MMP-2 expression enhances sensitivity to I-R injury, we performed unilateral I-R to induce mild tubular injury in wild-type mice. In contrast, expression of the NTT-MMP-2 isoform resulted in a dramatic increase in tubular cell necrosis, inflammation, and fibrosis. NTT-MMP-2 mice had enhanced expression of innate immunity genes and release of danger-associated molecular pattern molecules. We conclude that NTT-MMP-2 “primes” the kidney to enhanced susceptibility to I-R injury via induction of mitochondrial dysfunction. NTT-MMP-2 may be a novel AKI treatment target.

The alpha-1A-adrenergic receptor (AR) subtype is associated with cardioprotective signaling in the mouse and human heart. The rabbit is useful for cardiac disease modeling, but data on the alpha-1A in the rabbit heart are limited. Our objective was to test for expression and function of the alpha-1A in rabbit heart. By quantitative real-time reverse transcription PCR (qPCR) on mRNA from ventricular myocardium of adult male New Zealand White rabbits, the alpha-1B was 99% of total alpha-1-AR mRNA, with

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.

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 α1-adrenergic receptors (ARs). In RV trabeculae from nonfailing mouse hearts, α1-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, α1-ARs induced a positive inotropic response. Mechanistically, experiments with skinned trabeculae showed that α1-ARs decreased myofilament Ca2+ sensitivity in the nonfailing RV myocardium, whereas α1-ARs increased Ca2+ sensitivity in heart failure. This suggests that a switch in the Ca2+ sensitivity response to α1-AR stimulation explained the switch in the RV α1-AR inotropic response in heart failure. Myosin light chain kinase (MLCK) can increase myofilament Ca2+ 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 α1-AR inotropic response in heart failure. In the left ventricular myocardium, in contrast, α1-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 α1-ARs is switched from negative to positive in heart failure, through a pathway involving increased myofilament Ca2+ sensitivity. Since α1-AR agonist catecholamines are elevated in heart failure, increased α1-AR inotropic responses in the RV myocardium may be adaptive in heart failure by helping the failing RV respond to increased pulmonary pressures.

In beating hearts, phosphorylation of myosin regulatory light chain (RLC) at a single site to 0.45 mol of phosphate/mol by cardiac myosin light chain kinase (cMLCK) increases Ca(2+) sensitivity of myofilament contraction necessary for normal cardiac performance. Reduction of RLC phosphorylation in conditional cMLCK knock-out mice caused cardiac dilation and loss of cardiac performance by 1 week, as shown by increased left ventricular internal diameter at end-diastole and decreased fractional shortening. Decreased RLC phosphorylation by conventional or conditional cMLCK gene ablation did not affect troponin-I or myosin-binding protein-C phosphorylation in vivo. The extent of RLC phosphorylation was not changed by prolonged infusion of dobutamine or treatment with a β-adrenergic antagonist, suggesting that RLC is constitutively phosphorylated to maintain cardiac performance. Biochemical studies with myofilaments showed that RLC phosphorylation up to 90% was a random process. RLC is slowly dephosphorylated in both noncontracting hearts and isolated cardiac myocytes from adult mice. Electrically paced ventricular trabeculae restored RLC phosphorylation, which was increased to 0.91 mol of phosphate/mol of RLC with inhibition of myosin light chain phosphatase (MLCP). The two RLCs in each myosin appear to be readily available for phosphorylation by a soluble cMLCK, but MLCP activity limits the amount of constitutive RLC phosphorylation. MLCP with its regulatory subunit MYPT2 bound tightly to myofilaments was constitutively phosphorylated in beating hearts at a site that inhibits MLCP activity. Thus, the constitutive RLC phosphorylation is limited physiologically by low cMLCK activity in balance with low MLCP activity.

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 μm 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 ≈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.

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, P0.001). 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, P0.05); 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, P0.05), 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, P0.05; 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, P0.01). However, peak cytosolic [Ca(2+)] was not different (1.13 +/- 0.15 vs. 1.19 +/- 0.04 microM, n = 6-7, P0.05), 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.

Two functional α1-adrenergic receptor (AR) subtypes (α1Aand α1B) have been identified in the mouse heart. However, it is unclear whether the third known subtype, α1D-AR, is also present. To investigate this, we determined whether there were α1-AR responses in hearts from a novel mouse model lacking α1A- and α1B-ARs (double knockout) (ABKO). In Langendorff-perfused hearts, α1-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 α1D-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 α1-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 α1A- and α1B-ARs retain functional α1-AR responses involving decreases of coronary flow and ventricular pressure that reflect α1D-AR-mediated vasoconstriction. Furthermore, α1-AR inotropic responses depend critically on the experimental conditions.

D. T. McCloskey, D. G. Rokosh, T. D. O'Connell, E. C. Keung, P. C. Simpson and A. J. Baker. α1-Adrenoceptor Subtypes Mediate Negative Inotropy in Myocardium from α1A/C-Knockout and Wild Type Mice. Journal of Molecular and Cellular Cardiology (2002) 34, 1007–1017. Cardiac α1-adrenoceptors (AR) have two predominant subtypes (α1A-AR and α1B-AR) however, their roles in regulating contraction are unclear. We determined the effects of stimulating α1A-AR (using the subtype-selective agonist A61603) and α1B-AR (using a gene knockout mouse lacking α1A-AR) separately, and together (using phenylephrine) on Ca2+ transients, intracellular pH, and contraction of mouse cardiac trabeculae. Stimulation of α1-AR subtypes separately or together caused a triphasic contractile response. After a transient (≈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 α1A-AR plus α1B-AR stimulation (50±3%) was more than with separate subtype stimulation (P

Acute kidney injury (AKI) is a major risk factor for the development of chronic kidney disease (CKD). Persistent oxidative stress and mitochondrial dysfunction are implicated across diverse forms of AKI and in the transition to CKD. In this study, we applied hyperpolarized (HP) 13 C dehydroascorbate (DHA) and 13 C pyruvate magnetic resonance spectroscopy (MRS) to investigate the renal redox capacity and mitochondrial pyruvate dehydrogenase (PDH) activity, respectively, in a murine model of AKI at baseline and 7 days after unilateral ischemia reperfusion injury (IRI). Compared with the contralateral sham-operated kidneys, the kidneys subjected to IRI showed a significant decrease in the HP 13 C vitamin C/(vitamin C + DHA) ratio, consistent with a decrease in redox capacity. The kidneys subjected to IRI also showed a significant decrease in the HP 13 C bicarbonate/pyruvate ratio, consistent with impaired PDH activity. The IRI kidneys showed a significantly higher HP 13 C lactate/pyruvate ratio at day 7 compared with baseline, although the 13 C lactate/pyruvate ratio was not significantly different between the IRI and contralateral sham-operated kidneys at day 7. Arterial spin labeling magnetic resonance imaging (MRI) demonstrated significantly reduced perfusion in the IRI kidneys. Renal tissue analysis showed corresponding increased reactive oxygen species (ROS) and reduced PDH activity in the IRI kidneys. Our results show the feasibility of HP 13 C MRS for the non-invasive assessment of oxidative stress and mitochondrial PDH activity following renal IRI.

The full-length isoform of matrixmetalloproteinase-2 (FL-MMP-2) plays a role in turnover of the cardiac extracellular matrix. FL-MMP-2 is also present intracellularly in association with sarcomeres and, in the setting of oxidative stress, cleaves myofilament proteins with resultant impaired contractility. Recently, a novel N-terminal truncated MMP-2 isoform (NTT-MMP-2) generated during oxidative stress was identified and shown to induce severe systolic failure; however, the injury mechanisms remained unclear. In this study, cardiac-specific NTT-MMP-2 transgenic mice were used to determine the physiological effects of NTT-MMP-2 on: force development of intact myocardium; the function of cardiac myofilaments in demembranated myocardium; and on intracellular Ca2+ transients in isolated myocytes. We related the contractile defects arising from NTT-MMP-2 expression to the known intracellular locations of NTT-MMP-2 determined using immunohistochemistry. Comparison was made with the pathophysiology arising from cardiac-specific FL-MMP-2 transgenic mice. Consistent with previous studies, FL-MMP-2 was localized to myofilaments, while NTT-MMP-2 was concentrated within subsarcolemmal mitochondria and to sites in register with the Z-line. NTT-MMP-2 expression caused a 50% reduction of force development by intact myocardium. However, NTT-MMP-2 expression did not reduce myofilament force development, consistent with the lack of NTT-MMP-2 localization to myofilaments. NTT-MMP-2 expression caused a 50% reduction in the amplitude of Ca2+ transients, indicating impaired activation. Conclusions: Unlike FL-MMP-2, NTT-MMP-2 does not mediate myofilament damage. Instead, NTT-MMP-2 causes impaired myocyte activation, which may involve effects due to localization in mitochondria and/or to transverse tubules affecting Ca2+ transients. Thus, FL-MMP-2 and NTT-MMP-2 have discrete intracellular locations and mediate different intracellular damage to cardiac myocytes.

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.

Heart failure with reduced ejection fraction involves activation of the sympathetic nervous system and chronic hyperactivation of the sympatho-adrenergic receptors (ARs) β-ARs and α1-ARs, which are thought to be cardiotoxic and worsen pathological remodeling and function. Concurrently, the failing heart manifests significant decreases in sympathetic nerve terminal density, decreased cardiac norepinephrine levels, and marked downregulation of β-AR abundance and signaling. Thus, a state of both feast and famine coexist with respect to the adrenergic state in heart failure. For the failing heart, the hyperadrenergic state is toxic. However, the role of hypoadrenergic mechanisms in the pathophysiology of heart failure is less clear. Cardiotoxic effects are known to arise from the β1-AR subtype, and use of β-AR blockers is a cornerstone of current heart failure therapy. However, cardioprotective effects arise from the β2-AR subtype that counteract hyperactive β1-AR signaling, but unfortunately, β2-AR cardioprotective signaling in heart failure is inhibited by β-AR blocker therapy. In contrast to current dogma, recent research shows β1-AR signaling can also be cardioprotective. Moreover, for some forms of heart failure, β2-AR signaling is cardiotoxic. Thus for both β-AR subtypes, there is a balance between cardiotoxic versus cardioprotective effects. In heart failure, stimulation of α1-ARs is widely thought to be cardiotoxic. However, also contrary to current dogma, recent research shows that α1-AR signaling is cardioprotective. Taken together, recent research identifies cardioprotective signaling arising from β1-AR, β2-AR, and α1-ARs. A goal for future therapies will to harness the protective effects of AR signaling while minimizing cardiotoxic effects. The trajectory of heart failure therapy changed radically from the previous and intuitive use of sympathetic agonists, which unfortunately resulted in greater mortality, to the current use of β-AR blockers, which initially seemed counterintuitive. As a cautionary note, if the slow adoption of beta-blocker therapy in heart failure is any guide, then new treatment strategies, especially counterintuitive therapies involving stimulating β-AR and α1-AR signaling, may take considerable time to develop and gain acceptance.

In nonfailing mouse RV, α1-adrenergic receptors (α1-ARs) mediate a negative inotropic effect (NIE). We reported that α1-AR inotropy in heart failure was dramatically switched to a positive inotropic effect (PIE). The two predominant α1-AR subtypes in heart are the α1A and α1B. However, their inotropic roles in heart failure are unclear.Goal: Determine the roles of α1A and α1B subtypes in upregulation of α1-AR inotropy in failing RV.Methods: We used a mouse model of bleomycin-induced RV failure. Bleomycin or saline was instilled into the trachea. Using RV cardiac trabeculae, we assessed in-vitro α1-AR inotropic responses to non-subtype selective agonist phenylephrine (PE, stimulates α1A and α1B subtypes), or subtype-selective agonist A61603 (stimulates only α1A subtype).Results: Two wk after bleomycin, there was pulmonary fibrosis, pulmonary hypertension and RV failure. For non-failing RV, A61603 caused a NIE (force decreased 48±10%, n=3) similar to the NIE mediated by PE (force decreased 52±12%, n=3). Thus, stimulation of the α1A subtype singly, or together with the α1B subtype, produced a similar NIE. In contrast, for failing RV, stimulation with A61603 caused a switch to a PIE (force increased 134±36%, n=7; P

Cardiomyopathy is a major cause of morbidity and mortality. Ventricular conduction delay, as shown by prolonged deflections in the electrocardiogram caused by delayed ventricular contraction (wide QRS complex), is a common feature of cardiomyopathy and is associated with a poor prognosis. Although the G i -signaling pathway is up-regulated in certain cardiomyopathies, previous studies suggested this up-regulation was compensatory rather than a potential cause of the disease. Using the tetracycline transactivator system and a modified G i -coupled receptor (Ro1), we provide evidence that increased G i signaling in mice can result in a lethal cardiomyopathy associated with a wide QRS complex arrhythmia. Induced expression of Ro1 in adult mice resulted in a >90% mortality rate at 16 wk, whereas suppression of Ro1 expression after 8 wk protected mice from further mortality and allowed partial improvement in systolic function. Results of DNA-array analysis of over 6,000 genes from hearts expressing Ro1 are consistent with hyperactive G i signaling. DNA-array analysis also identified known markers of cardiomyopathy and hundreds of previously unknown potential diagnostic markers and therapeutic targets for this syndrome. Our system allows cardiomyopathy to be induced and reversed in adult mice, providing an unprecedented opportunity to dissect the role of G i signaling in causing cardiac pathology.

Compared to non-failing RV, we reported that in failing RV the inotropic response to stimulation of α1-adrenergic receptors (α1-ARs) was increased. Recently we found that a single α1-AR subtype (α1A) was responsible. However, the significance of increased α1A responses remains unclear.Goal: Determine if increased α1A-mediated inotropy is beneficial in RV failure and if the α1A-subtype is a potential therapeutic target.Methods: We used a RV-specific failure model in mice. Tracheal instillation of the fibrogenic antibiotic bleomycin induced pulmonary fibrosis, pulmonary hypertension, and RV failure within 2-3 wk. We assessed survival and investigated the cellular mechanisms contributing to increased α1A inotropy in failing RV. Furthermore, in the context of bleomycin induced RV failure, we determined if there was a beneficial effect of chronic (2 wk.) treatment of mice with the α1A agonist A61603 using osmotic mini-pump infusion.Results: For non-failing RV, acute α1A stimulation with A61603 induced a negative inotropic effect. In contrast, for failing RV, acute α1A stimulation induced a positive inotropic effect. A subset of animals did not manifest this switch in the inotropic response, and had significantly worse survival, suggesting that upregulation of the α1A inotropic response was beneficial in failing RV. Upregulation of α1A inotropy in failing RV was associated with increased Ca2+ transients, and increased phosphorylation of the myosin regulatory light chain (increased myofilament Ca2+ sensitivity). Moreover, consistent with a beneficial effect of increased α1A activation in failing RV, chronic treatment of mice with A61603 for 2 wk. resulted in less myocardial fibrosis, lower serum troponin level, and better in-vivo function assessed with echocardiography.Conclusion: Increased α1A subtype activation is beneficial in the failing RV. The α1A-subtype is a potential therapeutic target in failing RV.

Long-standing heavy alcohol consumption acts as a chronic stress on the heart. It is thought that alcohol-induced changes of contractility are due to altered Ca2+handling, but no measurements of cytosolic Ca2+([Ca2+]c) after chronic alcohol exposure have been made. Therefore experiments were performed to determine whether alcohol-induced changes in contractility are due to altered Ca2+handling by measuring [Ca2+]c(indo 1) in hearts from rats drinking 36% ethanol for 7 mo and age-matched controls. Peak left ventricular pressure was depressed (−16%), whereas rates of contraction (12%) and relaxation (14–20%) were faster in alcohol-exposed hearts. Systolic [Ca2+]c(808 ± 45 vs. 813 ± 45 nM), diastolic [Ca2+]c(195 ± 11 vs. 193 ± 10 nM), and rates of [Ca2+]crise and decline were the same in alcohol-exposed and control hearts. Protein levels of Ca2+-handling proteins, sarcoplasmic reticulum Ca2+-ATPase and phospholamban, were the same in myocytes isolated from alcohol-exposed and control hearts (SDS-polyacrylamide gel). These data suggest that chronic alcohol-induced contractile changes are not due to altered Ca2+handling but may be due to changes at the level of the myofilament. As a first step in elucidating the mechanism(s) of alcohol-induced changes at the myofilament, we assessed myosin heavy chain (MHC) isoform content (SDS-polyacrylamide gel). α-MHC was decreased relative to β-MHC ( a/ a+ b = 0.55 ± 0.03 vs. 0.66 ± 0.02; P < 0.02) in alcohol-exposed hearts, which cannot account for the observed alcohol-induced contractile changes. In conclusion, changes of myocardial contractility due to chronic alcohol exposure do not result from altered Ca2+handling but from changes at the level of the myofilament that do not involve MHC isoform shifts.

In this paper, we study the connectivity of multihop wireless networks with the log-normal shadowing model by investigating the critical transmission power required by each node for asymptotic vanishing of the isolated notes in the network. The vanishing of isolated nodes is not only a prerequisite but also a good indication of network connectivity. All previous known work on network connectivity under such a shadowing model were obtained only based on simulation studies or ignoring the important boundary effect to avoid the challenging technical analysis, and thus cannot be applied to any practical wireless networks. It is extremely challenging to take the complicated boundary effect into consideration under such a realistic model because the transmission area of each node is an irregular region other than a circular area. We assume the wireless nodes are represented by a Poisson point process with density n over a unit-area disk. With the complicated boundary effect taken into consideration, we first derive an explicit formula for the expected number of isolated nodes in the network. This explicit formula allows people to control the expected number of isolated nodes by tuning the node density or even the transmission power, and thus the desired level of network connectivity can be expected. Then we give an upper bound and a lower bound of the critical transmission power for asymptotic vanishing of the isolated nodes. Our upper and lower bounds for the critical transmission power used by each node are almost tight.

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.

A simple mathematical model describing the dynamic connection between Ca2+ and force generation in intact skeletal muscle from the frog has been developed from isometric force responses to cytosolic Ca2+ concentration ([Ca2+]c) transients during tetanic and twitch contractions. The main element of the model is a two-state cross-bridge cycle characterized by the fractional rate of cross-bridge attachment (f(app)) and the fractional rate of cross-bridge detachment (g*). While g* is constant, f(app) is time varying and regulated by both [Ca2+]c and force. Having only four adjustable parameters, the model is mathematically unique, thereby allowing precise parameter estimation from the dynamic Ca2+ and force data. The model should be useful for developing insights into the relative importance for force generation and relaxation of 1) the size and shape of the Ca2+ transient, 2) the sensitivity of the fractional rate of cross-bridge attachment to both the [Ca2+]c and the force responses, and 3) the fractional rate of cross-bridge detachment, which is insensitive to both Ca2+ and force.

Assessment of free cytosolic [Ca2+] ([Ca2+]c) using the acetoxymethyl ester (AM) form of indo-1 may be compromised by loading of indo-1 into noncytosolic compartments, primarily mitochondria. To determine the fraction of noncytosolic fluorescence in whole hearts loaded with indo-1 AM, Mn2+ was used to quench cytosolic fluorescence. Residual (i.e., noncytosolic) fluorescence was subtracted from the total fluorescence before calculating [Ca2+]c. Noncytosolic fluorescence was used to estimate mitochondrial [Ca2+]. In hearts paced at 5 Hz (N = 17), noncytosolic fluorescence was 0.61 +/- 0.06 and 0.56 +/- 0.07 of total fluorescence at lambda 385 and lambda 456, respectively. After taking into account noncytosolic fluorescence, systolic and diastolic [Ca2+]c was 673 +/- 72 and 132 +/- 9 nM, respectively, noncytosolic [Ca2+] was 183 +/- 36 nM and increased to 272 +/- 12 when extracellular Ca2+ was increased from 2 to 6 mM. This increase in noncytosolic [Ca2+] was inhibited by ruthenium red, a blocker of Ca2+ uptake by mitochondria. We conclude that cytosolic and mitochondrial [Ca2+] can be determined in whole hearts loaded with indo-1 AM by using Mn2+ to quench cytosolic fluorescence.

Electron paramagnetic resonance spectroscopy of a spin probe attached to cys-707 on myosin cross-bridges was used to monitor the orientation of the myosin catalytic domain at the beginning and end of the working power stroke in active muscle. Elevated concentrations of orthophosphate and decreased pH were used to shift the population of cross-bridges from force-producing states into low force, pre-power-stroke states. The spectrum of probes in active fibers was not changed by conditions that reduced tension by 70%, indicating that the orientation of the catalytic domain was the same at the beginning and end of the power stroke. Thus the data show that the catalytic domain remains rigidly oriented on the actin filament during the power stroke.

The goal of this study was to determine the effects of intracellular acidosis (pH approximately 6.3) of frog skeletal muscle on force and on intracellular Ca2+ concentration ([Ca2+]i; measured at 20 degrees C using indo 1 fluorescence). Acidosis reduced tetanic force by only 11 +/- 2% (mean +/- SE, n = 8) but increased tetanic [Ca2+]i by 33 +/- 6%, suggesting that acidosis reduced the maximum Ca(2+)-activated force. During relaxation, the [Ca2+]i at half-maximal force was doubled with acidosis, suggesting that acidosis altered the Ca(2+)-force relationship. Acidosis markedly slowed force relaxation and [Ca2+]i decline (time constants fitted to force and [Ca2+]i during relaxation increased by 133 +/- 20 and 68 +/- 13%, respectively, with acidosis), suggesting that slowed force relaxation with acidosis may arise from slowed Ca2+ clearance from the cytosol. Late in relaxation, at approximately 30% of initial force, there was a transient phase of [Ca2+]i increase that was delayed with acidosis in proportion to the slowing of force relaxation. This is consistent with previous suggestions that dissociation of cross-bridges from the thin filament during relaxation promotes Ca2+ release to the cytosol from troponin. This study concludes that in skeletal muscle acidosis has little effect on tetanic force and that the major effects are decreased Ca2+ sensitivity and slower relaxation.

Previous studies have shown considerable variability in the metabolic response of human skeletal muscle during a standardized exercise protocol. The goal of these studies was to investigate the factors responsible for the broad range of metabolic changes produced by fatiguing exercise. Experiments were performed to quantitate the measurement error of 31P nuclear magnetic resonance spectroscopy of human muscle, the reproducibility of changes within a single subject, and the effects of fasting, carbohydrate loading, and metabolic acidosis. The results show that none of these factors appear to be responsible for the wide variation between subjects. However, the effects of training and genetic factors were not investigated and are likely to be responsible for the substantial variability between subjects.© 1995 John Wiley &Sons, Inc.