Peter J. Reiser

Impaired Myofibrillar Energetics and Oxidative Injury During Human Atrial Fibrillation

Background—Atrial fibrillation (AF) is associated with severe contractile dysfunction and structural and electrophysiological remodeling. Mechanisms responsible for impaired contractility are undefined, and current therapies do not address this dysfunction. We have found that myofibrillar creatine kinase (MM-CK), an important controller of myocyte contractility, is highly sensitive to oxidative injury, and we hypothesized that increased oxidative stress and energetic impairment during AF could contribute to contractile dysfunction. Methods and Results—Right atrial appendages were obtained from AF patients undergoing the Maze procedure and from control patients who were in normal sinus rhythm and undergoing cardiac surgery. MM-CK activity was reduced in AF patients compared with controls (25.4±3.4 versus 18.2±3.8 &mgr;mol/mg of myofibrillar protein per minute; control versus AF;P <0.05). No reduction in total CK activity or myosin ATPase activity was detected. This selective reduction in MM-CK activity was associated with increased relative expression of the &bgr;-myosin isoform (25±6 versus 63±5%&bgr;, CTRL versus AF;P <0.05). Western blotting of AF myofibrillar isolates demonstrated no changes in protein composition but showed increased prevalence of protein oxidation as detected by Western blotting for 3-nitrotyrosine (peroxynitrite biomarker) and protein carbonyls (hydroxyl radical biomarker;P <0.05). Patterns of these oxidative markers were distinct, which suggests discrete chemical events and differential protein vulnerabilities in vivo. MM-CK inhibition was statistically correlated to extent of nitration (P <0.01) but not to carbonyl presence. Conclusions—The present results provide novel evidence of oxidative damage in human AF that altered myofibrillar energetics may contribute to atrial contractile dysfunction and that protein nitration may be an important participant in this condition.

Cancer cachexia is regulated by selective targeting of skeletal muscle gene products

Cachexia is a syndrome characterized by wasting of skeletal muscle and contributes to nearly one-third of all cancer deaths. Cytokines and tumor factors mediate wasting by suppressing muscle gene products, but exactly which products are targeted by these cachectic factors is not well understood. Because of their functional relevance to muscle architecture, such targets are presumed to represent myofibrillar proteins, but whether these proteins are regulated in a general or a selective manner is also unclear. Here we demonstrate, using in vitro and in vivo models of muscle wasting, that cachectic factors are remarkably selective in targeting myosin heavy chain. In myotubes and mouse muscles, TNF-alpha plus IFN-gamma strongly reduced myosin expression through an RNA-dependent mechanism. Likewise, colon-26 tumors in mice caused the selective reduction of this myofibrillar protein, and this reduction correlated with wasting. Under these conditions, however, loss of myosin was associated with the ubiquitin-dependent proteasome pathway, which suggests that mechanisms used to regulate the expression of muscle proteins may be cachectic factor specific. These results shed new light on cancer cachexia by revealing that wasting does not result from a general downregulation of muscle proteins but rather is highly selective as to which proteins are targeted during the wasting state.

TGF-β1 mediates the hypertrophic cardiomyocyte growth induced by angiotensin II

Angiotensin II (Ang II), a potent hypertrophic stimulus, causes significant increases in TGFb1 gene expression. However, it is not known whether there is a causal relationship between increased levels of TGF-beta1 and cardiac hypertrophy. Echocardiographic analysis revealed that TGF-beta1-deficient mice subjected to chronic subpressor doses of Ang II had no significant change in left ventricular (LV) mass and percent fractional shortening during Ang II treatment. In contrast, Ang II-treated wild-type mice showed a >20% increase in LV mass and impaired cardiac function. Cardiomyocyte cross-sectional area was also markedly increased in Ang II-treated wild-type mice but unchanged in Ang II-treated TGF-beta1-deficient mice. No significant levels of fibrosis, mitotic growth, or cytokine infiltration were detected in Ang II-treated mice. Atrial natriuretic factor expression was approximately 6-fold elevated in Ang II-treated wild-type, but not TGF-beta1-deficient mice. However, the alpha- to beta-myosin heavy chain switch did not occur in Ang II-treated mice, indicating that isoform switching is not obligatorily coupled with hypertrophy or TGF-beta1. The Ang II effect on hypertrophy was shown not to result from stimulation of the endogenous renin-angiotensis system. These results indicate that TGF-beta1 is an important mediator of the hypertrophic growth response of the heart to Ang II.

FIBROBLAST GROWTH FACTOR-2 MEDIATES PRESSURE-INDUCED HYPERTROPHIC RESPONSE

In vitro, fibroblast growth factor-2 (FGF2) has been implicated in cardiomyocyte growth and reexpression of fetal contractile genes, both markers of hypertrophy. However, its in vivo role in cardiac hypertrophy during pressure overload is not well characterized. Mice with or without FGF2 (Fgf2(+/+) and Fgf2(-/-), respectively) were subjected to transverse aortic coarctation (AC). Left ventricular (LV) mass and wall thickness were assessed by echocardiography preoperatively and once a week postoperatively for 10 weeks. In vivo LV function during dobutamine stimulation, cardiomyocyte cross-sectional area, and recapitulation of fetal cardiac genes were also measured. AC Fgf2(-/-) mice develop significantly less hypertrophy (4-24% increase) compared with AC Fgf2(+/+) mice (41-52% increase). Cardiomyocyte cross-sectional area is significantly reduced in AC Fgf2(-/-) mice. Noncoarcted (NC) and AC Fgf2(-/-) mice have similar beta-adrenergic responses, but those of AC Fgf2(+/+) mice are blunted. A lack of mitotic growth in both AC Fgf2(+/+) and Fgf2(-/-) hearts indicates a hypertrophic response of cardiomyocytes. Consequently, FGF2 plays a major role in cardiac hypertrophy. Comparison of alpha- and beta-cardiac myosin heavy chain mRNA and protein levels in NC and AC Fgf2(+/+) and Fgf2(-/-) mice indicates that myosin heavy chain composition depends on hemodynamic stress rather than on FGF2 or hypertrophy, and that isoform switching is transcriptionally, not posttranscriptionally, regulated.

Electrophoretic separation and quantitation of cardiac myosin heavy chain isoforms in eight mammalian species

A protocol for sample preparation and gel electrophoresis is described that reliably results in the separation of the alpha- and beta-isoforms of cardiac myosin heavy chain (MHC-alpha and MHC-beta) in eight mammalian species. The protocol is based on a simple, nongradient denaturing gel. The magnitude of separation of MHC-alpha and MHC-beta achieved with this protocol is sufficient for quantitative determination of the relative amounts of these two isoforms in mouse, rat, guinea pig, rabbit, canine, pig, baboon, and human myocardial samples. The sensitivity of the protocol is sufficient for the detection of MHC isoforms in samples at least as small as 1 microgram. The glycerol concentration in the separating gel is an important factor for successfully separating MHC-alpha and MHC-beta in myocardial samples from different species. The effect of sample load on MHC-alpha and MHC-beta band resolution is illustrated. The results also indicate that inclusion of a homogenization step during sample preparation increases the amount of MHC detected on the gel for cardiac samples to a much greater extent than for skeletal muscle samples. Although the protocol described in this study is excellent for analyzing cardiac samples, it should be noted that the same protocol is not optimal for separating MHC isoforms expressed in skeletal muscle, as is illustrated.A protocol for sample preparation and gel electrophoresis is described that reliably results in the separation of the α- and β-isoforms of cardiac myosin heavy chain (MHC-α and MHC-β) in eight mammalian species. The protocol is based on a simple, nongradient denaturing gel. The magnitude of separation of MHC-α and MHC-β achieved with this protocol is sufficient for quantitative determination of the relative amounts of these two isoforms in mouse, rat, guinea pig, rabbit, canine, pig, baboon, and human myocardial samples. The sensitivity of the protocol is sufficient for the detection of MHC isoforms in samples at least as small as 1 μg. The glycerol concentration in the separating gel is an important factor for successfully separating MHC-α and MHC-β in myocardial samples from different species. The effect of sample load on MHC-α and MHC-β band resolution is illustrated. The results also indicate that inclusion of a homogenization step during sample preparation increases the amount of MHC detected on the gel for cardiac samples to a much greater extent than for skeletal muscle samples. Although the protocol described in this study is excellent for analyzing cardiac samples, it should be noted that the same protocol is not optimal for separating MHC isoforms expressed in skeletal muscle, as is illustrated.

Ablation of sarcolipin enhances sarcoplasmic reticulum calcium transport and atrial contractility

Sarcolipin is a novel regulator of cardiac sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) and is expressed abundantly in atria. In this study we investigated the physiological significance of sarcolipin in the heart by generating a mouse model deficient for sarcolipin. The sarcolipin-null mice do not show any developmental abnormalities or any cardiac pathology. The absence of sarcolipin does not modify the expression level of other Ca2+ handling proteins, in particular phospholamban, and its phosphorylation status. Calcium uptake studies revealed that, in the atria, ablation of sarcolipin resulted in an increase in the affinity of the SERCA pump for Ca2+ and the maximum velocity of Ca2+ uptake rates. An important finding is that ablation of sarcolipin resulted in an increase in atrial Ca2+ transient amplitudes, and this resulted in enhanced atrial contractility. Furthermore, atria from sarcolipin-null mice showed a blunted response to isoproterenol stimulation, implicating sarcolipin as a mediator of β-adrenergic responses in atria. Our study documented that sarcolipin is a key regulator of SERCA2a in atria. Importantly, our data demonstrate the existence of distinct modulators for the SERCA pump in the atria and ventricles.

Intracellular distribution of peroxynitrite during doxorubicin cardiomyopathy: evidence for selective impairment of myofibrillar creatine kinase

Cardiac peroxynitrite and protein nitration are increased during doxorubicin cardiotoxicity, but the intracellular targets and functional consequences have not been defined. We investigated the intracellular distribution of protein nitration during doxorubicin cardiotoxicity in mice. Following in vivo cardiac function assessments by echocardiography, cardiac tissues were prepared for immunohistochemistry and electron microscopy 5 days after doxorubicin (20 mg kg−1) or vehicle control. Increased cardiac 3‐nitrotyrosine was observed using light microscopy in doxorubicin treated animals. Immunogold electron microscopy (55,000×) revealed increased myofibrillar and mitochondrial 3‐nitrotyrosine levels following doxorubicin, but cellular 3‐nitrotyrosine density was 2 fold higher in myofibrils. We therefore investigated the actions of peroxynitrite on intact cardiac contractile apparatus. Skinned ventricular trabeculae were exposed to physiologically relevant peroxynitrite concentrations (50 or 300 nM) for 1 h, then Ca2+ induced contractile responses were measured in the presence of ATP (4 mM) or phosphocreatine (12 mM) as high energy phosphate supplier. ATP maximal force generation was unaltered after 50 nM peroxynitrite, but phosphocreatine/ATP response was reduced (0.99±0.63 vs 1.59±0.11), suggesting selective inactivation of myofibrillar creatine kinase (MM‐CK). Reduction of ATP maximal force was observed at 300 nM peroxynitrite and phosphocreatine/ATP response was further reduced (0.64±0.30). Western blotting showed concentration dependent nitration of MM‐CK in treated trabeculae. Similarly, cardiac tissues from doxorubicin treated mice demonstrated increased nitration and inactivation of MM‐CK compared to controls. These results demonstrate that peroxynitrite‐related protein nitration are mechanistic events in doxorubicin cardiomyopathy and that the cardiac myofibril is an important oxidative target in this setting. Furthermore, MM‐CK may be a uniquely vulnerable target to peroxynitrite in vivo.

Differential effects of ovariectomy on calcium activation of cardiac and soleus myofilaments

The hypothesis that ovarian sex hormone deficiency affects cardiac myofilament activation was tested. Chemically skinned ventricular trabeculae and single soleus muscle fibers were prepared from 10- and 14-wk ovariectomized and control rats. Tension-pCa (-log [Ca2+]) relations of left ventricular trabeculae and soleus fibers were compared to test whether thin filament proteins are potential sites of modulated activation. Trabeculae from ovariectomized rats exhibited a significant increase in Ca2+ sensitivity with no change in maximal tension-generating ability. In contrast, soleus fibers demonstrated no shift in Ca2+ sensitivity but generated significantly less maximal tension. No changes in thin filament protein isoform expression or loss of thin filament proteins were apparent in the trabeculae or soleus fibers from ovariectomized rats. Although not directly tested, our results are consistent with a possible modulation of regulatory proteins (e.g., cardiac troponin I) to account for the observed change in myofilament responsiveness of hearts from ovariectomized rats. Other possible mechanisms for the altered myocardial Ca2+ sensitivity after ovariectomy are discussed.

Redox modulation of global phosphatase activity and protein phosphorylation in intact skeletal muscle

Skeletal muscles produce transient reactive oxygen species (ROS) in response to intense stimulation, disuse atrophy, heat stress, hypoxia, osmotic stress, stretch and cell receptor activation. The physiological significance is not well understood. Protein phosphatases (PPases) are known to be highly sensitive to oxidants and could contribute to many different signalling responses in muscle. We tested whether broad categories of PPases are inhibited by levels of acute oxidant exposure that do not result in loss of contractile function or gross oxidative stress. We also tested if this exposure results in elevated levels of global protein phosphorylation. Rat diaphragm muscles were treated with either 2,3‐dimethoxy‐1‐naphthoquinone (DMNQ; 1, 10, 100 μm; a mitochondrial O2•−/H2O2 generator) or exogenous H2O2 (5, 50, 500 μm) for 30 min. Supernatants were assayed for serine/threonine PPase (Ser/Thr‐PPase) or protein tyrosine PPase (PTP) activities. With the exception of 500 μm H2O2, no other oxidant exposures significantly elevated protein carbonyl formation, nor did they alter the magnitude of twitch force. DMNQ significantly decreased all categories of PPase activity at 10 and 100 μm and reduced PTP at 1 μm. Similar reductions in Ser/Thr‐PPase activity were seen in response to 50 and 500 μm H2O2 and PTP at 500 μm H2O2. ROS treatments resulted a dose‐dependent increase in the phosphorylation states of many proteins. The data are consistent with the concept that PPases, within intact skeletal muscles, are highly sensitive to acute changes in ROS activity and that localized ROS play a critical role in lowering the barriers for effective phosphorylation events to occur in muscle cells, thus increasing the probability for cell signalling responses to proceed.

Myosin light chain isoform expression among single mammalian skeletal muscle fibers: species variations.

Extensive heterogeneity in myosin heavy chain and light chain (MLC) isoform expression in skeletal muscle has been well documented in several mammalian species. The initial objective of this study was to determine the extent of heterogeneity in myosin isoform expression among single fibers in limb muscles of dogs, a species for which relatively little has been reported. Fibers were isolated from muscles that have different functions with respect to limb extension and limb flexion and were analyzed on SDS gels, with respect to myosin isoform composition. The results of this part of the study indicate that there are at least four distinct fiber types in dog limb and diaphragm muscles, on the basis of MLC isoform expression: conventional fast (expressing fast-type isoforms of MLC1 (MLC1F) and MLC2 (MLC2F), plus MLC3), conventional slow (expressing slow-type MLC1 (MLC1S) and MLC2 (MLC2S)), hybrid (expressing MLC1S, MLC1F, MLC2S, MLC2F and MLC3) and a second slow fiber type, designated as S1F. S1F fibers express MLC1F, along with MLC1S and MLC2S and relatively low levels of MLC3. The fraction of slow fibers that are S1F fibers varies among dog limb muscles, being greater in limb extensors than flexors. Furthermore, the mean level of MLC1F in S1F fibers is greater in extensors than flexors (mean levels range from ~3% to 50% of total MLC1). The study was, therefore, extended to include six additional species, spanning a broad range in adult body size to more thoroughly characterize heterogeneity in MLC isoform expression among mammals. The results indicate that there are distinct patterns in MLC isoform expression among fast and slow fibers among different species. Specifically, large-size mammals have two distinct types of slow fibers, based upon MLC isoform composition (conventional and S1F fibers), whereas small mammals exhibit variations in MLC isoforms between different types of fast fibers, including a fast fiber type that expresses MLC1S (designated as F1S fibers). S1F fibers were absent in rodent muscles and F1S fibers were not found in large mammals. We conclude that extensive variation exists in MLC isoform expression in mammalian skeletal muscle fibers, yet there are distinct patterns among different species and among muscles within an individual species.