scholarly journals Exacerbation of dystrophic cardiomyopathy by phospholamban deficiency mediated chronically increased cardiac Ca2+ cycling in vivo

2018 ◽  
Vol 315 (6) ◽  
pp. H1544-H1552 ◽  
Author(s):  
Michelle L. Law ◽  
Kurt W. Prins ◽  
Megan E. Olander ◽  
Joseph M. Metzger
Keyword(s):  
Cardiac Myocytes ◽  
Heart Function ◽  
Regulatory Protein ◽  
Membrane Damage ◽  
Mdx Mice ◽  
Blue Dye ◽  
Double Knockout ◽  
Intracellular Ca ◽  
Dystrophic Cardiomyopathy

Cardiomyopathy is a significant contributor to morbidity and mortality in Duchenne muscular dystrophy (DMD). Membrane instability, leading to intracellular Ca2+ mishandling and overload, causes myocyte death and subsequent fibrosis in DMD cardiomyopathy. On a cellular level, cardiac myocytes from mdx mice have dysregulated Ca2+ handling, including increased resting Ca2+ and slow Ca2+ decay, especially evident under stress conditions. Sarco(endo)plasmic reticulum Ca2+ ATPase and its regulatory protein phospholamban (PLN) are potential therapeutic targets for DMD cardiomyopathy owing to their key role in regulating intracellular Ca2+ cycling. We tested the hypothesis that enhanced cardiac Ca2+ cycling would remediate cardiomyopathy caused by dystrophin deficiency. We used a genetic complementation model approach by crossing dystrophin-deficient mdx mice with PLN knockout (PLNKO) mice [termed double-knockout (DKO) mice]. As expected, adult cardiac myocytes isolated from DKO mice exhibited increased contractility and faster relaxation associated with increased Ca2+ transient peak height and faster Ca2+ decay rate compared with control mice. However, compared with wild-type, mdx, and PLNKO mice, DKO mice unexpectedly had reduced in vivo systolic and diastolic function as measured by echocardiography. Furthermore, Evans blue dye uptake was increased in DKO hearts compared with control, mdx, and PLNKO hearts, demonstrating increased membrane damage, which subsequently led to increased fibrosis in the DKO myocardium in vivo. In conclusion, despite enhanced intracellular Ca2+ handling at the myocyte level, DMD cardiomyopathy was exacerbated owing to unregulated chronic increases in Ca2+ cycling in DKO mice in vivo. These findings have potentially important implications for ongoing therapeutic strategies for the dystrophic heart. NEW & NOTEWORTHY This study examined the effects of phospholamban ablation on the pathophysiology of cardiomyopathy in dystrophin-deficient mice. In this setting, contractility and Ca2+ cycling were enhanced in isolated myocytes; however, in vivo heart function was diminished. Additionally, sarcolemmal integrity was compromised and fibrosis was increased. This is the first study, to our knowledge, examining unregulated Ca2+ cycling in the dystrophin-deficient heart. Results from this study have implications for potential therapies targeting Ca2+ handling in dystrophic cardiomyopathy. Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/unregulated-ca2-cycling-exacerbates-dmd-cardiomyopathy/ .

Inspiratory loading does not accelerate dystrophy inmdx mouse diaphragm: implications for regenerative therapy

2003 ◽  
Vol 94 (2) ◽  
pp. 411-419 ◽  
Author(s):  
Alexander S. Krupnick ◽  
Jianliang Zhu ◽  
Taitan Nguyen ◽  
Daniel Kreisel ◽  
Keki R. Balsara ◽  
...  
Keyword(s):  
Therapeutic Strategy ◽  
Mdx Mouse ◽  
Mdx Mice ◽  
Blue Dye ◽  
Regenerative Therapy ◽  
Regenerative Capacity ◽  
Progressive Degeneration ◽  
Mouse Diaphragm ◽  
The Relationship

Since the finding that the mdx mouse diaphragm, in contrast to limb muscles, undergoes progressive degeneration analogous to that seen in Duchenne muscular dystrophy, the relationship between the workload on a muscle and the pathogenesis of dystrophy has remained controversial. We increased the work performed by the mdx mouse diaphragm in vivo by tracheal banding and evaluated the progression of dystrophic changes in that muscle. Despite the establishment of dramatically increased respiratory workload and accelerated myofiber damage documented by Evans blue dye, no change in the pace of progression of dystrophy was seen in banded animals vs. unbanded, sham-operated controls. At the completion of the study, more centrally nucleated fibers were evident in the diaphragms of banded mdx mice than in sham-operated mdx controls, indicating that myofiber regeneration increases to meet the demands of the work-induced damage. These data suggest that there is untapped regenerative capacity in dystrophin-deficient muscle and validates experimental efforts aimed at augmenting regeneration within skeletal muscle as a therapeutic strategy in the treatment of dystrophinopathies.

Cellular and functional defects in a mouse model of heart failure

2000 ◽  
Vol 279 (6) ◽  
pp. H3101-H3112 ◽  
Author(s):  
Giovanni Esposito ◽  
L. F. Santana ◽  
Keith Dilly ◽  
Jader Dos Santos Cruz ◽  
Lan Mao ◽  
...  
Keyword(s):  
Heart Failure ◽  
Adrenergic Receptor ◽  
Heart Function ◽  
Imaging Techniques ◽  
Single Cells ◽  
Structural Protein ◽  
Muscle Lim Protein ◽  
Intracellular Ca ◽  
Whole Heart

Heart failure and dilated cardiomyopathy develop in mice that lack the muscle LIM protein (MLP) gene (MLP−/−). The character and extent of the heart failure that occurs in MLP−/− mice were investigated using echocardiography and in vivo pressure-volume (P-V) loop measurements. P-V loop data were obtained with a new method for mice (sonomicrometry) using two pairs of orthogonal piezoelectric crystals implanted in the endocardial wall. Sonomicrometry revealed right-shifted P-V loops in MLP−/−mice, depressed systolic contractility, and additional evidence of heart failure. Cellular changes in MLP−/− mice were examined in isolated single cells using patch-clamp and confocal Ca2+ concentration ([Ca2+]) imaging techniques. This cellular investigation revealed unchanged Ca2+ currents and Ca2+ spark characteristics but decreased intracellular [Ca2+] transients and contractile responses and a defect in excitation-contraction coupling. Normal cellular and whole heart function was restored in MLP−/− mice that express a cardiac-targeted transgene, which blocks the function of β-adrenergic receptor (β-AR) kinase-1 (βARK1). These data suggest that, despite the persistent stimulus to develop heart failure in MLP−/− mice (i.e., loss of the structural protein MLP), downregulation and desensitization of the β-ARs may play a pivotal role in the pathogenesis. Furthermore, this work suggests that the inhibition of βARK1 action may prove an effective therapy for heart failure.

scholarly journals TAT-μUtrophin mitigates the pathophysiology of dystrophin and utrophin double-knockout mice

2011 ◽  
Vol 111 (1) ◽  
pp. 200-205 ◽  
Author(s):  
Jarrod A. Call ◽  
James M. Ervasti ◽  
Dawn A. Lowe
Keyword(s):  
Knockout Mice ◽  
Skeletal Muscles ◽  
Ex Vivo ◽  
Eccentric Contraction ◽  
Mdx Mice ◽  
Double Knockout ◽  
Replacement Treatment ◽  
Generating Capacity

Previously, we demonstrated functional substitution of dystrophin by TAT-μUtrophin (TAT-μUtr) in dystrophin-deficient mdx mice. Herein, we addressed whether TAT-μUtr could improve the phenotype of dystrophin and utrophin double-knockout ( mdx:utr−/−) mice. Specifically, we quantitatively compared survival and quality of life assessments in mdx:utr−/− mice receiving TAT-μUtr protein administration against placebo-treated mdx:utr−/− mice (PBS). Additionally, skeletal muscles from TAT-μUtr and PBS mice were tested in vivo and ex vivo for strength and susceptibility to eccentric contraction-induced injury. We found the TAT-μUtr treatment extended life span 45% compared with mice administered PBS. This was attributed to significantly increased food consumption (3.1 vs. 1.8 g/24 h) due to improved ability to search for food as daily cage activities were greater in TAT-μUtr mice (e.g., 364 vs. 201 m ambulation/24 h). The extensor digitorum longus muscles of TAT-μUtr-treated double-knockout mice also displayed increased force-generating capacity ex vivo (8.3 vs. 6.4 N/cm2) and decreased susceptibility to injury ex vivo and in vivo. These data indicate that the functional benefits of TAT-μUtr replacement treatment extend to the mdx:utr−/− double-knockout mouse and support its development as a therapy to mitigate muscle weakness in patients with Duchenne muscular dystrophy.

Abstract 8: GTPase-Activating Protein for Rho Associated with FAK (GRAF) Regulates Cardiomyocyte Membrane Integrity

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Thomas J O'Neill ◽  
Kaitlin Lenhart ◽  
Jason Doherty ◽  
Mauricio Rojas ◽  
Mack P Christopher ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Membrane Damage ◽  
Membrane Integrity ◽  
Muscle Pathology ◽  
Blue Dye ◽  
Membrane Repair ◽  
Cell Membrane Damage ◽  
Deficient Mice

Cardiac myocytes are unique in their requirement to sustain continuous repetitive contraction in the setting of intense mechanical stress while simultaneously maintaining high membrane integrity for an appropriate electrical gradient. The consequence of failure of the membrane repair response has been highlighted in recent reports linking cardiomyocyte membrane fragility with cardiac degeneration in patients as well as in their analogous mouse models. Herein, we describe a novel role for GTPase activator for Rho associated with Focal Adhesion Kinase (GRAF) in regulating cardiomyocyte membrane integrity. We previously published that disruption of GRAF in Xenopus laevis resulted in progressive skeletal muscle degeneration. We now show that GRAF-depleted tadpoles exhibit defective cardiac formation and function. Interestingly, damage of muscle cells in vivo and in vitro led to a translocation of GRAF to the sarcolemma, suggesting that GRAF may be an important component of the cardiac membrane repair machinery. To further explore this possibility, we generated GRAF hypomorphic mice that exhibit greater than 99% reduction of endogenous GRAF expression. While GRAF deficient mice show normal Mendelian birth distribution and are viable, they exhibit a modest skeletal muscle pathology. Although baseline cardiac integrity was not compromised in GRAF deficient mice, treatment either with cardiotoxin or intraperitoneal injection of isoproterenol led to elevated cardiomyocyte membrane damage (assessed by Evan’s blue dye uptake) in GRAF deficient compared to control mice (19% vs 2% of myocytes within afflicted ventricular area for cardiotoxin, 18% vs 8% for isoproterenol respectively). Moreover, cultured GRAF null myocytes exhibited a significantly attenuated membrane resealing response following laser-mediated disruption compared to GRAF-containing control cells as assessed by accumulation of the membrane impermeable dye, FM-143. As well, the survival rate after injury of GRAF-deficient cells was markedly attenuated (20% vs 85% in control cells). While cardiac cell membrane damage is likely a frequent and important event, the repair process is currently understudied, and this is the first report to implicate a Rho regulator in this response.

scholarly journals Deficient BH4 production via de novo and salvage pathways regulates NO responses to cytokines in adult cardiac myocytes

2008 ◽  
Vol 295 (5) ◽  
pp. H2178-H2187 ◽  
Author(s):  
Irina A. Ionova ◽  
Jeannette Vásquez-Vivar ◽  
Jennifer Whitsett ◽  
Anja Herrnreiter ◽  
Meetha Medhora ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
De Novo ◽  
Regulatory Protein ◽  
Cell Types ◽  
No Production ◽  
Inos Protein ◽  
Protein Dimers ◽  
Adult Cardiac Myocytes ◽  
Salvage Pathways

Adult rat cardiac myocytes typically display a phenotypic response to cytokines manifested by low or no increases in nitric oxide (NO) production via inducible NO synthase (iNOS) that distinguishes them from other cell types. To better characterize this response, we examined the expression of tetrahydrobiopterin (BH4)-synthesizing and arginine-utilizing genes in cytokine-stimulated adult cardiac myocytes. Intracellular BH4 and 7,8-dihydrobiopterin (BH2) and NO production were quantified. Cytokines induced GTP cyclohydrolase and its feedback regulatory protein but with deficient levels of BH4 synthesis. Despite the induction of iNOS protein, cytokine-stimulated adult cardiac myocytes produced little or no increase in NO versus unstimulated cells. Western blot analysis under nonreducing conditions revealed the presence of iNOS monomers. Supplementation with sepiapterin (a precursor of BH4) increased BH4 as well as BH2, but this did not enhance NO levels or eliminate iNOS monomers. Similar findings were confirmed in vivo after treatment of rat cardiac allograft recipients with sepiapterin. It was found that expression of dihydrofolate reductase, required for full activity of the salvage pathway, was not detected in adult cardiac myocytes. Thus, adult cardiac myocytes have a limited capacity to synthesize BH4 after cytokine stimulation. The mechanisms involve posttranslational factors impairing de novo and salvage pathways. These conditions are unable to support active iNOS protein dimers necessary for NO production. These findings raise significant new questions about the prevailing understanding of how cytokines, via iNOS, cause cardiac dysfunction and injury in vivo during cardiac inflammatory disease states since cardiac myocytes are not a major source of high NO production.

scholarly journals Acute Detubulation of Ventricular Myocytes Amplifies the Inhibitory Effect of Cholinergic Agonist on Intracellular Ca2+ Transients

2021 ◽  
Vol 12 ◽  
Author(s):  
Andriy E. Belevych ◽  
Vladimir Bogdanov ◽  
Dmitry A. Terentyev ◽  
Sandor Gyorke
Keyword(s):  
Muscarinic Receptor ◽  
Cardiac Myocytes ◽  
Muscarinic Receptors ◽  
Parasympathetic Nervous System ◽  
Ventricular Myocytes ◽  
Heart Function ◽  
Critical Role ◽  
Inhibitory Effect ◽  
Intracellular Ca ◽  
Image Pixels

Muscarinic receptors expressed in cardiac myocytes play a critical role in the regulation of heart function by the parasympathetic nervous system. How the structural organization of cardiac myocytes affects the regulation of Ca2+ handling by muscarinic receptors is not well-defined. Using confocal Ca2+ imaging, patch-clamp techniques, and immunocytochemistry, the relationship between t-tubule density and cholinergic regulation of intracellular Ca2+ in normal murine ventricular myocytes and myocytes with acute disruption of the t-tubule system caused by formamide treatment was studied. The inhibitory effect of muscarinic receptor agonist carbachol (CCh, 10 μM) on the amplitude of Ca2+ transients, evoked by field-stimulation in the presence of 100 nM isoproterenol (Iso), a β-adrenergic agonist, was directly proportional to the level of myocyte detubulation. The timing of the maximal rate of fluorescence increase of fluo-4, a Ca2+-sensitive dye, was used to classify image pixels into the regions functionally coupled or uncoupled to the sarcolemmal Ca2+ influx (ICa). CCh decreased the fraction of coupled regions and suppressed Ca2+ propagation from sarcolemma inside the cell. Formamide treatment reduced ICa density and decreased sarcoplasmic reticulum (SR) Ca2+ content. CCh did not change SR Ca2+ content in Iso-stimulated control and formamide-treated myocytes. CCh inhibited peak ICa recorded in the presence of Iso by ∼20% in both the control and detubulated myocytes. Reducing ICa amplitude up to 40% by changing the voltage step levels from 0 to –25 mV decreased Ca2+ transients in formamide-treated but not in control myocytes in the presence of Iso. CCh inhibited CaMKII activity, whereas CaMKII inhibition with KN93 mimicked the effect of CCh on Ca2+ transients in formamide-treated myocytes. It was concluded that the downregulation of t-tubules coupled with the diminished efficiency of excitation–contraction coupling, increases the sensitivity of Ca2+ release and propagation to muscarinic receptor-mediated inhibition of both ICa and CaMKII activity.

scholarly journals L-type channel inactivation balances the increased peak calcium current due to absence of Rad in cardiomyocytes

2021 ◽  
Vol 153 (9) ◽  
Author(s):  
Brooke M. Ahern ◽  
Andrea Sebastian ◽  
Bryana M. Levitan ◽  
Jensen Goh ◽  
Douglas A. Andres ◽  
...  
Keyword(s):  
Calcium Current ◽  
Knockout Mouse ◽  
Heart Function ◽  
Systolic Function ◽  
Cardiac Contraction ◽  
Calcium Flux ◽  
Double Knockout ◽  
Calcium Dependent ◽  
Dependent Inactivation

The L-type Ca2+ channel (LTCC) provides trigger calcium to initiate cardiac contraction in a graded fashion that is regulated by L-type calcium current (ICa,L) amplitude and kinetics. Inactivation of LTCC is controlled to fine-tune calcium flux and is governed by voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). Rad is a monomeric G protein that regulates ICa,L and has recently been shown to be critical to β-adrenergic receptor (β-AR) modulation of ICa,L. Our previous work showed that cardiomyocyte-specific Rad knockout (cRadKO) resulted in elevated systolic function, underpinned by an increase in peak ICa,L, but without pathological remodeling. Here, we sought to test whether Rad-depleted LTCC contributes to the fight-or-flight response independently of β-AR function, resulting in ICa,L kinetic modifications to homeostatically balance cardiomyocyte function. We recorded whole-cell ICa,L from ventricular cardiomyocytes from inducible cRadKO and control (CTRL) mice. The kinetics of ICa,L stimulated with isoproterenol in CTRL cardiomyocytes were indistinguishable from those of unstimulated cRadKO cardiomyocytes. CDI and VDI are both enhanced in cRadKO cardiomyocytes without differences in action potential duration or QT interval. To confirm that Rad loss modulates LTCC independently of β-AR stimulation, we crossed a β1,β2-AR double-knockout mouse with cRadKO, resulting in a Rad-inducible triple-knockout mouse. Deletion of Rad in cardiomyocytes that do not express β1,β2-AR still yielded modulated ICa,L and elevated basal heart function. Thus, in the absence of Rad, increased Ca2+ influx is homeostatically balanced by accelerated CDI and VDI. Our results indicate that the absence of Rad can modulate the LTCC without contribution of β1,β2-AR signaling and that Rad deletion supersedes β-AR signaling to the LTCC to enhance in vivo heart function.

scholarly journals Sarcolipin haploinsufficiency prevents dystrophic cardiomyopathy in mdx mice

2021 ◽  
Vol 320 (1) ◽  
pp. H200-H210 ◽  
Author(s):  
Satvik Mareedu ◽  
Ronald Pachon ◽  
Jayapalraj Thilagavathi ◽  
Nadezhda Fefelova ◽  
Rekha Balakrishnan ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Diastolic Dysfunction ◽  
Signaling Pathways ◽  
Cardiac Contractility ◽  
Mdx Mice ◽  
Intracellular Ca ◽  
Dystrophic Cardiomyopathy

First, reducing sarcopolin (SLN) expression improves sarco/endoplasmic reticulum Ca2+ uptake and intracellular Ca2+ handling and prevents cardiomyopathy in mdx mice. Second, reducing SLN expression prevents diastolic dysfunction and improves cardiac contractility in mdx mice Third, reducing SLN expression activates apelin-mediated cardioprotective signaling pathways in mdx heart.

scholarly journals GATA4 is a survival factor in adult cardiac myocytes but is not required for α1A-adrenergic receptor survival signaling

2008 ◽  
Vol 295 (2) ◽  
pp. H699-H707 ◽  
Author(s):  
Yuan Huang ◽  
Casey D. Wright ◽  
Satoru Kobayashi ◽  
Chastity L. Healy ◽  
Megan Elgethun ◽  
...  
Keyword(s):  
Cell Death ◽  
Cardiac Myocytes ◽  
Adrenergic Receptor ◽  
Double Knockout ◽  
Survival Factor ◽  
Downstream Effector ◽  
Adult Cardiac Myocytes ◽  
Survival Signaling ◽  
Downstream Mediator

Recently, we defined an α1A-adrenergic receptor-ERK (α1A-AR-ERK) survival signaling pathway in adult cardiac myocytes. Previous studies in neonatal cardiac myocytes indicated that the cardiac-specific transcription factor GATA4 is a downstream mediator of α1-ERK signaling and that phosphorylation of GATA4 by ERK increases DNA binding and transcriptional activity. Therefore, we examined GATA4 as a potential downstream effector of α1A-ERK survival signaling in adult cardiac myocytes. We measured norepinephrine (NE)-induced cell death in cultured cardiac myocytes lacking α1-ARs (cultured from α1A/B-AR double-knockout mice, α1ABKO mice) that are susceptible to cell death induced by several proapoptotic stimuli, including NE. Our results show that overexpression of GATA4 is sufficient to protect α1ABKO cardiac myocytes from NE-induced cell death. However, we found that the α1A-subtype did not induce phosphorylation or increase the activity of GATA4 in adult mouse cardiac myocytes in culture or in vivo. Furthermore, we examined the effect of siRNA-mediated knockdown of GATA4 on α1A-survival signaling. In α1B-knockout cardiac myocytes, which express only the α1A-subtype and are protected from NE-induced cell death, GATA4 knockdown did not reverse α1A-survival signaling in response to NE. In summary, we found that GATA4 acted as a survival factor by preventing cell death in α1ABKO cardiac myocytes, but GATA4 was not activated by α1-AR stimulation and was not required for α1A-survival signaling in adult cardiac myocytes. This also identifies an important mechanistic difference in α1-signaling between adult and neonatal cardiac myocytes.

PAF increases vascular permeability without increasing pulmonary arterial pressure in the rat

2001 ◽  
Vol 90 (1) ◽  
pp. 261-268 ◽  
Author(s):  
Leonardo C. Clavijo ◽  
Mary B. Carter ◽  
Paul J. Matheson ◽  
Mark A. Wilson ◽  
William B. Wead ◽  
...  
Keyword(s):  
Arterial Pressure ◽  
Pulmonary Edema ◽  
Pulmonary Arterial Pressure ◽  
Ex Vivo ◽  
Platelet Activating Factor ◽  
Microvascular Permeability ◽  
Blue Dye ◽  
E Coli ◽  
Pulmonary Arterial

In vivo pulmonary arterial catheterization was used to determine the mechanism by which platelet-activating factor (PAF) produces pulmonary edema in rats. PAF induces pulmonary edema by increasing pulmonary microvascular permeability (PMP) without changing the pulmonary pressure gradient. Rats were cannulated for measurement of pulmonary arterial pressure (Ppa) and mean arterial pressure. PMP was determined by using either in vivo fluorescent videomicroscopy or the ex vivo Evans blue dye technique. WEB 2086 was administered intravenously (IV) to antagonize specific PAF effects. Three experiments were performed: 1) IV PAF, 2) topical PAF, and 3) Escherichia coli bacteremia. IV PAF induced systemic hypotension with a decrease in Ppa. PMP increased after IV PAF in a dose-related manner. Topical PAF increased PMP but decreased Ppa only at high doses. Both PMP (88 ± 5%) and Ppa (50 ± 3%) increased during E. coli bacteremia. PAF-receptor blockade prevents changes in Ppa and PMP after both topical PAF and E. coli bacteremia. PAF, which has been shown to mediate pulmonary edema in prior studies, appears to act in the lung by primarily increasing microvascular permeability. The presence of PAF might be prerequisite for pulmonary vascular constriction during gram-negative bacteremia.

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