Biomedical subjects

Jeffrey Robbins

Publications and source records attributed to Jeffrey Robbins.

At least 19 recordsLinked to original sources

PubMed2006

Heart failure and protein quality control.

The heart is constantly under mechanical, metabolic, and thermal stress, even at baseline physiological conditions, and cardiac stress may increase as a result of environmental or intrinsic pathological insults. Cardiomyocytes are continuously challenged to efficiently and properly fold nascent polypeptides, traffic them to their appropriate cellular locations, and keep them from denaturing in the face of normal and pathological stimuli. Because deployment of misfolded or unfolded proteins can be disastrous, cells, in general, and cardiomyocytes, in particular, have developed a multilayered protein quality control system for maintaining proper protein conformation and for reorganizing and removing misfolded or aggregated polypeptides. Here, we examine recent data pointing to the importance of protein quality control in cardiac homeostasis and disease.

Animals↗

PubMed2006

Regulated overexpression of the A1-adenosine receptor in mice results in adverse but reversible changes in cardiac morphology and function.

BACKGROUND: Both the A1- and A3-adenosine receptors (ARs) have been implicated in mediating the cardioprotective effects of adenosine. Paradoxically, overexpression of both A1-AR and A3-AR is associated with changes in the cardiac phenotype. To evaluate the temporal relationship between AR signaling and cardiac remodeling, we studied the effects of controlled overexpression of the A1-AR using a cardiac-specific and tetracycline-transactivating factor-regulated promoter. METHODS AND RESULTS: Constitutive A1-AR overexpression caused the development of cardiac dilatation and death within 6 to 12 weeks. These mice developed diminished ventricular function and decreased heart rate. In contrast, when A1-AR expression was delayed until 3 weeks of age, mice remained phenotypically normal at 6 weeks, and >90% of the mice survived at 30 weeks. However, late induction of A1-AR still caused mild cardiomyopathy at older ages (20 weeks) and accelerated cardiac hypertrophy and the development of dilatation after pressure overload. These changes were accompanied by gene expression changes associated with cardiomyopathy and fibrosis and by decreased Akt phosphorylation. Discontinuation of A1-AR induction mitigated cardiac dysfunction and significantly improved survival rate. CONCLUSIONS: These data suggest that robust constitutive myocardial A1-AR overexpression induces a dilated cardiomyopathy, whereas delaying A1-AR expression until adulthood ameliorated but did not eliminate the development of cardiac pathology. Thus, the inducible A1-AR transgenic mouse model provides novel insights into the role of adenosine signaling in heart failure and illustrates the potentially deleterious consequences of selective versus nonselective activation of adenosine-signaling pathways in the heart.

Animals↗

PubMed2006

Cardiac myosin binding protein C phosphorylation is cardioprotective.

Cardiac myosin binding protein C (cMyBP-C) has three phosphorylatable serines at its N terminus (Ser-273, Ser-282, and Ser-302), and the residues' phosphorylation states may alter thick filament structure and function. To examine the effects of cMyBP-C phosphorylation, we generated transgenic mice with cardiac-specific expression of a cMyBP-C in which the three phosphorylation sites were mutated to aspartic acid, mimicking constitutive phosphorylation (cMyBP-C(AllP+)). The allele was bred into a cMyBP-C null background (cMyBP-C((t/t))) to ensure the absence of endogenous dephosphorylated cMyBP-C. cMyBP-C(AllP+) was incorporated normally into the cardiac sarcomere and restored normal cardiac function in the null background. However, subtle changes in sarcomere ultrastructure, characterized by increased distances between the thick filaments, indicated that phosphomimetic cMyBP-C affects thick-thin filament relationships, and yeast two-hybrid data and pull-down studies both showed that charged residues in these positions effectively prevented interaction with the myosin heavy chain. Confirming the physiological relevance of these data, the cMyBP-C(AllP+:(t/t)) hearts were resistant to ischemia-reperfusion injury. These data demonstrate that cMyBP-C phosphorylation functions in maintaining thick filament spacing and structure and can help protect the myocardium from ischemic injury.

Amino Acid Sequence↗

PubMed2006

Shifts in the myosin heavy chain isozymes in the mouse heart result in increased energy efficiency.

Cardiac-specific transgenesis in the mouse is widely used to study the basic biology and chemistry of the heart and to model human cardiovascular disease. A fundamental difference between mouse and human hearts is the background motor protein: mouse hearts contain predominantly the alphaalpha-myosin heavy chain (MyHC) isozyme while human hearts contain predominantly the betabeta-MyHC isozyme. Although the intrinsic differences in mechanical and enzymatic properties of the alphaalpha- and betabeta-MyHC molecules are well known, the consequences of isozyme shifts on energetics of the intact beating heart remain unknown. Therefore, we compared the free energy of ATP hydrolysis (|DeltaG( approximately ATP)|) determined by (31)P-NMR spectroscopy in isolated perfused littermate mouse hearts containing the same amount of myosin comprised of either >95% alphaalpha-MyHC or approximately 83% betabeta-MyHC. |DeltaG( approximately ATP)| was approximately 2 kJ mol(-1) higher in the betabeta-MyHC hearts at all workloads. Furthermore, upon inotropic challenge, hearts containing predominantly betabeta-MyHC hearts increased developed pressure more than alphaalpha-MyHC hearts whereas heart rate increased more in alphaalpha-MyHC hearts. Thus, hearts containing predominantly the betabeta-MyHC isozyme are more energy efficient than alphaalpha-MyHC hearts. We suggest that these fundamental differences in the motor protein energy efficiency at the whole heart level should be considered when interpreting results using mouse-based cardiovascular modeling of normal and diseased human hearts.

Adenosine Triphosphate↗

PubMed2006

PKC-betaII sensitizes cardiac myofilaments to Ca2+ by phosphorylating troponin I on threonine-144.

Ventricular myocytes express Galphaq-coupled receptors that can mediate enhanced contractility by increasing the sensitivity of the contractile apparatus to Ca(2+). The precise mechanisms underlying this change have been difficult to define, in part because myofilament regulatory proteins contain multiple phosphorylation sites for protein kinase C (PKC), protein kinase A (PKA) and myosin light chain kinase (MLCK), with potentially opposing effects. MLCK increases whereas PKC and PKA have a strong tendency to decrease myofilament Ca(2+) sensitivity in myocardium. Here we show in mouse cardiac myocytes that PKC-betaII can increase Ca(2+) sensitivity of tension by a similar magnitude to MLCK but via a distinct mechanism. For PKC-betaII (32)P-incorporation occurred primarily into cardiac troponin I (cTnI) and functional effects were highly dependent upon mutations in phosphorylation sites of cTnI. Replacement of serines-23/24 (PKA sites) with alanine prevented cross-phosphorylation of these sites, reduced (32)P-incorporation into cTnI by half and resulted in myofilament Ca(2+) sensitization rather than desensitization in response to PKC-betaII. Replacement of three additional sites on cTnI, serines-43/45 and threonine-144, eliminated PKC-betaII-mediated Ca(2+) sensitization and the remaining (32)P-incorporation into cTnI. A preference for PKC-betaII phosphorylation of threonine-144 in the intact filament lattice was revealed by differential stable isotope labeling and supported by an analysis of peptide phosphorylation. The results suggest that threonine-144 within the critical inhibitory domain of cTnI represents a novel site of regulation of myofilament Ca(2+) sensitivity by PKC-betaII, with possible implications for chronically stressed or diseased hearts.

Actin Cytoskeleton↗

PubMed2006

The effect of myosin regulatory light chain phosphorylation on the frequency-dependent regulation of cardiac function.

Although it has been suggested that in cardiac muscle the phosphorylation level of myosin regulatory light chain (RLC) correlates with frequency of stimulation, its significance in the modulation of the force-frequency and pressure-frequency relationships remains unclear. We examined the role of RLC phosphorylation on the force-frequency relation (papillary muscles), the pressure-frequency relation (Langendorff perfused hearts) and shortening-frequency relation (isolated cardiac myocytes) in nontransgenic (NTG) and transgenic mouse hearts expressing a nonphosphorylatable RLC protein (RLC(P-)). At 22 degrees C, NTG and RLC(P-) muscles showed a negative force-frequency relation. At 32 degrees C, at frequencies above 1 Hz, both groups showed a flat force-frequency relation. There was a small increase in RLC phosphorylation in NTG muscles when the frequency of stimulation was increased from 0.2 Hz to 4.0 Hz. However, the level of RLC phosphorylation in these isolated muscles was significantly lower compared to samples taken from NTG intact hearts. In perfused hearts, there was no difference in the slope of pressure-frequency relationship between groups, but the RLC(P-) group consistently developed a reduced systolic pressure and demonstrated a decreased contractility. There was no difference in the level of RLC phosphorylation in hearts paced at 300 and 600 bpm. In RLC(P-) hearts, the level of TnI phosphorylation was reduced compared to NTG. There was no change in the expression of PLB between groups, but expression of SERCA2 was increased in hearts from RLC(P-) compared to NTG. In isolated cardiac myocytes, there was no change in shortening-frequency relationship between groups. Moreover, there was no change in Ca(2+) transient parameters in cells from NTG and RLC(P-) hearts. Our data demonstrate that in cardiac muscle RLC phosphorylation is not an essential determinant of force- and pressure-frequency relations but the absence of RLC phosphorylation decreases contractility in force/pressure developing preparations.

Animals↗

PubMed2006

Neural crest cells retain multipotential characteristics in the developing valves and label the cardiac conduction system.

Multipotent neural crest cells (NCCs) are a major extracardiac component of cardiovascular development. Although recognized as contributing cells to the arterial valves at early developmental stages, NCC persistence in the valves at later times or in the adult heart is controversial. We analyzed NCC persistence and contributions to both semilunar and atrioventricular (AV) valves in the mature heart. Two NCC-specific promoters driving Cre recombinase, Wnt1-Cre and P0-Cre, were mated with floxed reporter mice, R26R or CAG-CAT-EGFP, to map NCC fate. Hearts were analyzed before aorticopulmonary (AP) septation through adult stages. As previously demonstrated, strong NCC labeling was detected in ventral and dorsal outflow cushions before AP septation. In contrast to previous reports, we found that substantial numbers of labeled cells persisted in the semilunar valves in late fetal, neonatal, and adult hearts. Furthermore, NCCs were also found in the AV valves, almost exclusively in the septal leaflets. NCCs in the AV valves expressed melanocytic and neurogenic markers. However, cells labeled in the proximal cardiac conduction system exhibited neurogenic and gliagenic markers, whereas some NCCs expressed no differentiation specific markers. These results suggest that cardiac NCCs contribute to the mature valves and the cardiac conduction system and retain multipotent characteristics late in development.

Aging↗

PubMed2006

Overexpressed cardiac Gsalpha in rabbits.

We overexpressed cardiac Gsalpha in rabbits using the beta-myosin heavy chain promoter. Gsalpha protein levels in the heart were increased 3-fold by Western blotting in both juvenile (3-4 months), adult (8-10 months), and older (11-16 months) rabbits, compared with wild type (WT) littermates. In transgenic (TG) rabbits, baseline levels of heart rate were elevated, P<0.05 (268+/-17 vs. 209+/-15 beats/min), as well as left ventricular (LV) contractility (LV dP/dt 5475+/-482 vs. 3740+/-246 mm Hg/s). These values and LV ejection fraction remained significantly elevated in older TG rabbits (11-16 months). However, maximal levels of LV dP/dt and heart rate with a high dose of isoproterenol (0.4 microg/kg/min) were similar in adult TG and WT rabbits. In isolated myocytes from the LV of adult rabbits, baseline percent contraction was increased, P<0.05, in TG (11.2+/-0.5%) compared to WT (9.3+/-0.5%), while maximal responses to isoproterenol (100 nM) were similar in adult TG (16.2+/-0.5%) and WT myocytes (15.6+/-0.4%). Although TG mice with overexpressed cardiac Gsalpha develop cardiomyopathy at 8-12 months of age, even at 16 months of age, there was no evidence of cardiomyopathy either in terms of LV function or histology in TG rabbits. In addition, Gialpha was elevated in the LV of adult (8-10 months old) TG rabbits compared to WT, but not in juvenile (3-5 months old) TG rabbits. Although both TG mice and rabbits with overexpressed cardiac Gsalpha exhibited enhanced heart rate and contractility, the TG rabbit does not develop cardiomyopathy, potentially due to a compensatory increase in Gialpha.

Adenylyl Cyclases↗

PubMed2006

Imaging cellular signals in the heart in vivo: Cardiac expression of the high-signal Ca2+ indicator GCaMP2.

Genetically encoded sensor proteins provide unique opportunities to advance the understanding of complex cellular interactions in physiologically relevant contexts; however, previously described sensors have proved to be of limited use to report cell signaling in vivo in mammals. Here, we describe an improved Ca(2+) sensor, GCaMP2, its inducible expression in the mouse heart, and its use to examine signaling in heart cells in vivo. The high brightness and stability of GCaMP2 enable the measurement of myocyte Ca(2+) transients in all regions of the beating mouse heart and prolonged pacing and mapping studies in isolated, perfused hearts. Transgene expression is efficiently temporally regulated in cardiomyocyte GCaMP2 mice, allowing recording of in vivo signals 4 weeks after transgene induction. High-resolution imaging of Ca(2+) waves in GCaMP2-expressing embryos revealed key aspects of electrical conduction in the preseptated heart. At embryonic day (e.d.) 10.5, atrial and ventricular conduction occur rapidly, consistent with the early formation of specialized conduction pathways. However, conduction is markedly slowed through the atrioventricular canal in the e.d. 10.5 heart, forming the basis for an effective atrioventricular delay before development of the AV node, as rapid ventricular activation occurs after activation of the distal AV canal tissue. Consistent with the elimination of the inner AV canal muscle layer at e.d. 13.5, atrioventricular conduction through the canal was abolished at this stage. These studies demonstrate that GCaMP2 will have broad utility in the dissection of numerous complex cellular interactions in mammals, in vivo.

Amino Acid Sequence↗

PubMed2006

Changes in end-to-end interactions of tropomyosin affect mouse cardiac muscle dynamics.

The ends of striated muscle tropomyosin (TM) are integral for thin filament cooperativity, determining the cooperative unit size and regulating the affinity of TM for actin. We hypothesized that altering the alpha-TM carboxy terminal overlap end to the beta-TM counterpart would affect the amino-terminal association, which would alter the end-to-end interactions of TM molecules in the thin filament regulatory strand and affect the mechanisms of cardiac muscle contraction. To test this hypothesis, we generated transgenic (TG) mouse lines that express a mutant form of alpha-TM in which the first 275 residues are from alpha-TM and the last nine amino acids are from beta-TM (alpha-TM9aaDeltabeta). Molecular analyses show that endogenous alpha-TM mRNA and protein are nearly completely replaced with alpha-TM9aaDeltabeta. Working heart preparations data show that the rates of contraction and relaxation are reduced in alpha-TM9aaDeltabeta hearts. Left ventricular pressure and time to peak pressure are also reduced (-12% and -13%, respectively). The ratio of maximum to minimum first derivatives of change in left ventricular systolic pressure with respect to time (ratio of +dP/dt to -dP/dt, respectively) is increased, but tau is not changed significantly. Force-intracellular calcium concentration ([Ca2+]i) measurements from intact papillary fibers demonstrate that alpha-TM9aaDeltabeta TG fibers produce less force per given [Ca2+]i compared with nontransgenic fibers. Taken together, the data demonstrate that the rate of contraction is primarily affected in TM TG hearts. Protein docking studies show that in the mutant molecule, the overall carbon backbone is perturbed about 1.5 A, indicating that end-to-end interactions are altered. These results demonstrate that the localized flexibility present in the coiled-coil structures of TM isoforms is different, and that plays an important role in interacting with neighboring thin filament regulatory proteins and with differentially modulating the myofilament activation processes.

Actin Cytoskeleton↗

PubMed2005

Mitochondrial dysfunction and apoptosis underlie the pathogenic process in alpha-B-crystallin desmin-related cardiomyopathy.

BACKGROUND: Mitochondria and sarcomeres have a well-defined architectural relation that partially depends on the integrity of the cytoskeletal network. An R120G missense mutation in the small heat shock protein alpha-B-crystallin (CryAB) causes desmin-related cardiomyopathy. Desmin-related cardiomyopathy is characterized by the formation of intracellular aggregates containing CryAB and desmin that are amyloid positive, and disease can be recapitulated in transgenic mice by cardiac-specific expression of the mutant protein. METHODS AND RESULTS: To understand the resultant pathology, we explored the acute effects of R120G expression both in vitro and in vivo. In vitro, transfection of adult cardiomyocytes with R120G-expressing adenovirus resulted in altered contractile mechanics. In vivo, as the cytoskeletal network is disturbed but before deficits in organ function can be detected, alterations in mitochondrial organization and architecture occur, leading to a reduction in the maximal rate of oxygen consumption with substrates that utilize complex I activity, alterations in the permeability transition pore, and compromised inner membrane potential. Apoptotic pathways are subsequently activated, which eventually results in cardiomyocyte death, dilation, and heart failure. CONCLUSIONS: Cardiac chaperone dysfunction acutely leads to altered cardiomyocyte mechanics, perturbations in mitochondrial-sarcomere architecture, and deficits in mitochondrial function, which can result in activation of apoptosis and heart failure.

Amyloid↗

PubMed2005

Cardiac myosin-binding protein-C phosphorylation and cardiac function.

The role of cardiac myosin binding protein-C (cMyBP-C) phosphorylation in cardiac physiology or pathophysiology is unclear. To investigate the status of cMyBP-C phosphorylation in vivo, we determined its phosphorylation state in stressed and unstressed mouse hearts. cMyBP-C phosphorylation is significantly decreased during the development of heart failure or pathologic hypertrophy. We then generated transgenic (TG) mice in which the phosphorylation sites of cMyBP-C were changed to nonphosphorylatable alanines (MyBP-C(AllP-)). A TG line showing &40% replacement with MyBP-C(AllP-) showed no changes in morbidity or mortality but displayed depressed cardiac contractility, altered sarcomeric structure and upregulation of transcripts associated with a hypertrophic response. To explore the effect of complete replacement of endogenous cMyBP-C with MyBP-C(AllP-), the mice were bred into the MyBP-C(t/t) background, in which less than 10% of normal levels of a truncated MyBP-C are present. Although MyBP-C(AllP-) was incorporated into the sarcomere and expressed at normal levels, the mutant protein could not rescue the MyBP-C(t/t) phenotype. The mice developed significant cardiac hypertrophy with myofibrillar disarray and fibrosis, similar to what was observed in the MyBP-C(t/t) animals. In contrast, when the MyBP-C(t/t) mice were bred to a TG line expressing normal MyBP-C (MyBP-CWT), the MyBP-C(t/t) phenotype was rescued. These data suggest that cMyBP-C phosphorylation is essential for normal cardiac function.

Animals↗

PubMed2005

Noonan syndrome mutation Q79R in Shp2 increases proliferation of valve primordia mesenchymal cells via extracellular signal-regulated kinase 1/2 signaling.

The molecular pathways regulating valve development are only partially understood. Recent studies indicate that dysregulation of mitogen-activated protein kinase (MAPK) signaling might play a major role in the pathogenesis of congenital valvular malformations, and, in this study, we explored the role of extracellular signal-regulated kinase (ERK) 1/2 activation in valve primordia expressing the Noonan syndrome mutation Q79R-Shp2. Noonan syndrome is an autosomal dominant disease characterized by dysmorphic features and cardiac abnormalities, with frequent pulmonic stenosis. The Q79R mutation of PTPN11 previously identified in Noonan syndrome families results in a gain-of-function of the encoded protein tyrosine phosphatase Shp2. We compared the effects of wild-type Shp2 and Q79R-Shp2 on endocardial cushion development. Atrioventricular and outflow tract endocardial cushions were excised from chick embryos, infected with wild-type Shp2 or Q79R-Shp2 adenovirus and embedded in a gel matrix. Q79R-Shp2, but not wild-type-Shp2, expression resulted in increased outgrowth of cells into the gel. The dependence of the Q79R-Shp2 effect on ERK1/2 and p38 MAPK signaling was then determined. The MAPK/ERK kinase (MEK)-1 inhibitor U0126, but not the p38-MAPK pathway inhibitor SB203580, abolished the effect of Q79R-Shp2 on cushion outgrowth. Coinfection with Q79R-Shp2 and dominant negative MEK-1 prevented enhanced endocardial cushion outgrowth, whereas expression of constitutively active MEK-1 mimicked the effect of Q79R-Shp2. Furthermore, dissociated cushion cells displayed increased 5-bromodeoxyuridine incorporation when infected with Q79R-Shp2 but not with wild-type Shp2. This promitotic effect was eliminated by U0126. Our results demonstrate that ERK1/2 activation is both necessary and sufficient to mediate the hyperproliferative effect of a gain-of-function mutation of Shp2 on mesenchymal cells in valve primordia.

Animals↗

PubMed2005

Reversal of amyloid-induced heart disease in desmin-related cardiomyopathy.

Amyloid oligomers, similar to the toxic entities found in Alzheimer's disease patients and in other amyloid-based diseases, are present in cardiomyocytes derived from human heart-failure patients and in animal models of desmin-related cardiomyopathy (DRM). The R120G mutation in alpha-B-crystallin (CryAB) causes DRM and is characterized by aggresomes containing CryAB(R120G) and amyloid oligomer. In this study, we show that aggresome levels do not correlate with disease. Blocking aggresome formation results in increased levels of toxic amyloid oligomer and decreased cardiomyocyte viability. We confirmed the primary toxicity of intrasarcoplasmic amyloid accumulation in the cardiomyocytes by ectopic expression of the amyloidogenic peptide PQ81, which consists of multiple repeats of a polyglutamine tract. We then addressed the issue of disease reversibility by placing CryAB(R120G) under inducible cardiomyocyte-specific expression in transgenic mice. The mice developed aggresomes and contained high concentrations of amyloid oligomer in the heart, resulting in cardiac disease. Cessation of CryAB(R120G) expression in symptomatic mice improved cardiac function and rescued all of the animals from premature death. Rescue was accompanied by significant decreases in amyloid oligomer without a significant reduction in aggresomes. Blocking cardiac amyloid oligomer formation, even after cardiac dysfunction presents, may be a therapeutic strategy in DRM as well as in other types of cardiac disease in which significant amyloid accumulation occurs.

Amino Acid Substitution↗

PubMed2005

Nuclear import of {alpha}B-crystallin is phosphorylation-dependent and hampered by hyperphosphorylation of the myopathy-related mutant R120G.

Phosphorylation modulates the functioning of alphaB-crystallin as a molecular chaperone. We here explore the role of phosphorylation in the nuclear import and cellular localization of alphaB-crystallin in HeLa cells. Inhibition of nuclear export demonstrated that phosphorylation of alphaB-crystallin is required for import into the nucleus. As revealed by mutant analysis, phosphorylation at Ser-59 is crucial for nuclear import, and phosphorylation at Ser-45 is required for speckle localization. Co-immunoprecipitation experiments suggested that the import of alphaB-crystallin is possibly regulated by its phosphorylation-dependent interaction with the survival motor neuron (SMN) protein, an important factor in small nuclear ribonucleoprotein nuclear import and assembly. This interaction was supported by co-localization of endogenous phosphorylated alphaB-crystallin with SMN in nuclear structures. The cardiomyopathy-causing alphaB-crystallin mutant R120G was found to be excessively phosphorylated, which disturbed SMN interaction and nuclear import, and resulted in the formation of cytoplasmic inclusions. Like for other protein aggregation disorders, hyperphosphorylation appears as an important aspect of the pathogenicity of alphaB-crystallin R120G.

Active Transport, Cell Nucleus↗

PubMed2005

Remodeling of gap junctions and slow conduction in a mouse model of desmin-related cardiomyopathy.

OBJECTIVE: We studied a transgenic mouse model of human desmin-related cardiomyopathy with cardiac-specific expression of a 7-amino acid deletion mutation in desmin (D7-des) to test the hypothesis that impaired linkage between desmin and desmosomes alters expression and function of the electrical coupling protein, connexin43 (Cx43). METHODS: Expression of Cx43 and selected mechanical junctions proteins was characterized in left ventrices of D7-des and control mice by quantitative confocal microscopy and immunoblotting. Remodeling of gap junctions was also analyzed by electron microscopic morphometry. The electrophysiological phentoype of D7-des mice was characterized by electrocardiography and optical mapping of transmembrane voltage. RESULTS: Cx43 signal at intercalated disks was decreased by approximately 3-fold in D7-des ventricular tissue due to reductions in both gap junction number and size. Immunoreactive signal at cell-cell junctions was also reduced significantly for adhesion molecules and linker proteins of desmosomes and fascia adherens junctions. Electron microscopy showed decreased gap junction remodeling. However, immunoblotting showed that the total tissue content of Cx43 and mechanical junction proteins was not reduced, suggesting that diminished signal at cell-cell junctions was not due to insufficient protein expression, but to failure of these proteins to assemble properly within electrical and mechanical junctions. Remodeling of gap junctions in D7-des mice led to slowing of ventricular conduction as demonstrated by optical electrophysiological mapping. CONCLUSIONS: These results illustrate how a defect in a protein conventionally thought to fulfill a mechanical function in the heart can also lead to electrophysiological alterations that may contribute to arrhythmogenesis.

Animals↗

PubMed2005

The essential light chain N-terminal extension alters force and fiber kinetics in mouse cardiac muscle.

The functional significance of the actin-binding region at the N terminus of the cardiac myosin essential light chain (ELC) remains elusive. In a previous experiment, the endogenous ventricular ELC was replaced with a protein containing a 10-amino acid deletion at positions 5-14 (ELC1vDelta5-14, referred to as 1vDelta5-14), a region that interacts with actin. 1vDelta5-14 mice showed no discernable mutant phenotype in skinned ventricular strips. However, because the myofilament lattice swells upon skinning, the mutant phenotype may have been concealed by the inability of the ELC to reach the actin-binding site. Using the same mouse model, we repeated earlier measurements and performed additional experiments on skinned strips osmotically compressed to the intact lattice spacing as determined by x-ray diffraction. 1vDelta5-14 mice exhibited decreased maximum isometric tension without a change in calcium sensitivity. The decreased force was most evident in 5-6-month-old mice compared with 13-15-month-old mice and may account for the greater ventricular wall thickness in young 1vDelta5-14 mice compared with age-matched controls. No differences were observed in unloaded shortening velocity at maximum calcium activation. However, 1vDelta5-14 mice exhibited a significant difference in the frequency at which minimum complex modulus amplitude occurred, indicating a change in cross-bridge kinetics. We hypothesize that the ELC N-terminal extension interaction with actin inhibits the reversal of the power stroke, thereby increasing isometric force. Our results strongly suggest that an interaction between residues 5-14 of the ELC N terminus and the C-terminal residues of actin enhances cardiac performance.

Actin Cytoskeleton↗

PubMed2005

Forced expression of alpha-myosin heavy chain in the rabbit ventricle results in cardioprotection under cardiomyopathic conditions.

BACKGROUND: The biochemical differences between the 2 mammalian cardiac myosin heavy chains (MHCs), alpha-MHC and beta-MHC, are well described, but the physiological consequences of basal isoform expression and isoform shifts in response to altered cardiac load are not clearly understood. Mature human ventricle contains primarily the beta-MHC isoform. However, the alpha-MHC isoform can be detected in healthy human ventricle and appears to be significantly downregulated in failing hearts. The unique biochemical properties of the alpha-MHC isoform might offer functional advantages in a failing heart that is expressing only the beta-MHC isoform. This hypothesis cannot be tested in mice or rats because both species express alpha-MHC as the predominant isoform. METHODS AND RESULTS: To test the effects of persistent alpha-MHC expression on the background of beta-MHC, we made transgenic (TG) rabbits that expressed rabbit alpha-MHC cDNA in the ventricle so that the endogenous myosin was partially replaced by the transgenically encoded species. Molecular, histological, and functional analyses showed no significant baseline effects in the TG rabbits compared with nontransgenic (NTG) littermates. To determine whether alpha-MHC expression afforded any advantages to stressed myocardium, a cohort of TG and NTG rabbits was subjected to rapid ventricular pacing. Although both the TG and NTG rabbits developed dilated cardiomyopathy, the TG rabbits had a higher shortening fraction, less septal thinning, and more normal +/-dP/dt than paced NTG rabbits. CONCLUSIONS: Transgenic expression of alpha-MHC does not have any apparent detrimental effects under basal conditions and is cardioprotective in experimental tachycardia-induced cardiomyopathy.

Animals↗