In cardiac muscle, the intercalated disk (ID) at the longitudinal cell-edges of cardiomyocytes provides as a macromolecular infrastructure that integrates mechanical and electrical coupling within ...the heart. Pathophysiological disturbance in composition of this complex is well known to trigger cardiac arrhythmias and pump failure. The mechanisms underlying assembly of this important cellular domain in human heart is currently unknown.
We collected 18 specimens from individuals that died from non-cardiovascular causes. Age of the specimens ranged from a gestational age of 15 weeks through 11 years postnatal. Immunohistochemical labeling was performed against proteins comprising desmosomes, adherens junctions, the cardiac sodium channel and gap junctions to visualize spatiotemporal alterations in subcellular location of the proteins.
Changes in spatiotemporal localization of the adherens junction proteins (N-cadherin and ZO-1) and desmosomal proteins (plakoglobin, desmoplakin and plakophilin-2) were identical in all subsequent ages studied. After an initial period of diffuse and lateral labelling, all proteins were fully localized in the ID at approximately 1 year after birth. Nav1.5 that composes the cardiac sodium channel and the gap junction protein Cx43 follow a similar pattern but their arrival in the ID is detected at (much) later stages (two years for Nav1.5 and seven years for Cx43, respectively).
Our data on developmental maturation of the ID in human heart indicate that generation of the mechanical junctions at the ID precedes that of the electrical junctions with a significant difference in time. In addition arrival of the electrical junctions (Nav1.5 and Cx43) is not uniform since sodium channels localize much earlier than gap junction channels.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
N
-Methyladenosine (m6A) methylation is the most prevalent internal posttranscriptional modification on mammalian mRNA. The role of m6A mRNA methylation in the heart is not known.
To determine the ...role of m6A methylation in the heart, we isolated primary cardiomyocytes and performed m6A immunoprecipitation followed by RNA sequencing. We then generated genetic tools to modulate m6A levels in cardiomyocytes by manipulating the levels of the m6A RNA methylase methyltransferase-like 3 (METTL3) both in culture and in vivo. We generated cardiac-restricted gain- and loss-of-function mouse models to allow assessment of the METTL3-m6A pathway in cardiac homeostasis and function.
We measured the level of m6A methylation on cardiomyocyte mRNA, and found a significant increase in response to hypertrophic stimulation, suggesting a potential role for m6A methylation in the development of cardiomyocyte hypertrophy. Analysis of m6A methylation showed significant enrichment in genes that regulate kinases and intracellular signaling pathways. Inhibition of METTL3 completely abrogated the ability of cardiomyocytes to undergo hypertrophy when stimulated to grow, whereas increased expression of the m6A RNA methylase METTL3 was sufficient to promote cardiomyocyte hypertrophy both in vitro and in vivo. Finally, cardiac-specific METTL3 knockout mice exhibit morphological and functional signs of heart failure with aging and stress, showing the necessity of RNA methylation for the maintenance of cardiac homeostasis.
Our study identified METTL3-mediated methylation of mRNA on N
-adenosines as a dynamic modification that is enhanced in response to hypertrophic stimuli and is necessary for a normal hypertrophic response in cardiomyocytes. Enhanced m6A RNA methylation results in compensated cardiac hypertrophy, whereas diminished m6A drives eccentric cardiomyocyte remodeling and dysfunction, highlighting the critical importance of this novel stress-response mechanism in the heart for maintaining normal cardiac function.
Understanding relationships between heart failure and arrhythmias, important causes of suffering and sudden death, remains an unmet goal for biomedical researchers and physicians. Evidence assembled ...over the past decade supports a view that activation of the multifunctional Ca and calmodulin-dependent protein kinase II (CaMKII) favors myocardial dysfunction and cell membrane electrical instability. CaMKII activation follows increases in intracellular Ca or oxidation, upstream signals with the capacity to transition CaMKII into a Ca and calmodulin-independent constitutively active enzyme. Constitutively active CaMKII appears poised to participate in disease pathways by catalyzing the phosphorylation of classes of protein targets important for excitation–contraction coupling and cell survival, including ion channels and Ca homeostatic proteins, and transcription factors that drive hypertrophic and inflammatory gene expression. This rich diversity of downstream targets helps to explain the potential for CaMKII to simultaneously affect mechanical and electrical properties of heart muscle cells. Proof-of-concept studies from a growing number of investigators show that CaMKII inhibition is beneficial for improving myocardial performance and for reducing arrhythmias. We review the molecular physiology of CaMKII and discuss CaMKII actions at key cellular targets and results of animal models of myocardial hypertrophy, dysfunction, and arrhythmias that suggest CaMKII inhibition may benefit myocardial function while reducing arrhythmias.
Heart failure remains a major health burden around the world. Despite great progress in delineation of molecular mechanisms underlying development of disease, standard therapy has not advanced at the ...same pace. The multifunctional signaling molecule Ca
/calmodulin-dependent protein kinase II (CaMKII) has received considerable attention over recent years for its central role in maladaptive remodeling and arrhythmias in the setting of chronic disease. However, these basic science discoveries have yet to translate into new therapies for human patients. This review addresses both the promise and barriers to developing translational therapies that target CaMKII signaling to abrogate pathologic remodeling in the setting of chronic disease. Efforts in small molecule design are discussed, as well as alternative targeting approaches that exploit novel avenues for compound delivery and/or genetic approaches to affect cardiac CaMKII signaling. These alternative strategies provide hope for overcoming some of the challenges that have limited the development of new therapies.
Here, we present a novel computational model to study the effects of late Na
+
current (
I
Na,L
) in human atrial myocytes. Simulations predict that
I
Na,L
promotes intracellular accumulation of Ca
...2+
, with subsequent dysregulation of Ca
2+
/calmodulin-dependent protein kinase II (CaMKII) signaling and ryanodine receptor 2-mediated Ca
2+
release. Although
I
Na,L
plays a small role in regulating atrial myocyte excitability at baseline, CaMKII-dependent enhancement of the current promoted arrhythmogenic dynamics.
Atrial fibrillation (AF) affects more than three million people per year in the United States and is associated with high morbidity and mortality. Both electrical and structural remodeling contribute to AF, but the molecular pathways underlying AF pathogenesis are not well understood. Recently, a role for Ca
2+
/calmodulin-dependent protein kinase II (CaMKII) in the regulation of persistent “late” Na
+
current (
I
Na,L
) has been identified. Although
I
Na,L
inhibition is emerging as a potential antiarrhythmic strategy in patients with AF, little is known about the mechanism linking
I
Na,L
to atrial arrhythmogenesis. A computational approach was used to test the hypothesis that increased CaMKII-activated
I
Na,L
in atrial myocytes disrupts Ca
2+
homeostasis, promoting arrhythmogenic afterdepolarizations. Dynamic CaMKII activity and regulation of multiple downstream targets
I
Na,L
, L-type Ca
2+
current, phospholamban, and the ryanodine receptor sarcoplasmic reticulum Ca
2+
-release channel (RyR2) were incorporated into an existing well-validated computational model of the human atrial action potential. Model simulations showed that constitutive CaMKII-dependent phosphorylation of Na
v
1.5 and the subsequent increase in
I
Na,L
effectively disrupt intracellular atrial myocyte ion homeostasis and CaMKII signaling. Specifically, increased
I
Na,L
promotes intracellular Ca
2+
overload via forward-mode Na
+
/Ca
2+
exchange activity, which greatly increases RyR2 open probability beyond that observed for CaMKII-dependent phosphorylation of RyR2 alone. Increased
I
Na,L
promotes atrial myocyte repolarization defects (afterdepolarizations and alternans) in the setting of acute β-adrenergic stimulation. We anticipate that our modeling efforts will help identify new mechanisms for atrial Na
V
1.5 regulation with direct relevance for human AF.
NEW & NOTEWORTHY
Here, we present a novel computational model to study the effects of late Na
+
current (
I
Na,L
) in human atrial myocytes. Simulations predict that
I
Na,L
promotes intracellular accumulation of Ca
2+
, with subsequent dysregulation of Ca
2+
/calmodulin-dependent protein kinase II (CaMKII) signaling and ryanodine receptor 2-mediated Ca
2+
release. Although
I
Na,L
plays a small role in regulating atrial myocyte excitability at baseline, CaMKII-dependent enhancement of the current promoted arrhythmogenic dynamics.
Listen to this article’s corresponding podcast at
http://ajpheart.podbean.com/e/camkii-dependent-regulation-of-atrial-late-sodium-current-and-excitability/
.
Role of CaMKII in cardiac arrhythmias Hund, Thomas J., PhD; Mohler, Peter J., PhD
Trends in cardiovascular medicine,
07/2015, Letnik:
25, Številka:
5
Journal Article
Recenzirano
Odprti dostop
Abstract Protein phosphorylation is a central mechanism in vertebrates for the regulation of signaling. With regard to the cardiovascular system, phosphorylation of myocyte targets is critical for ...the regulation of excitation contraction coupling, metabolism, intracellular calcium regulation, mitochondrial activity, transcriptional regulation, and cytoskeletal dynamics. In fact, pathways that tune protein kinase signaling have been a mainstay for cardiovascular therapies for the past 60 years. The calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine kinase with numerous roles in human physiology. Dysfunction in CaMKII-based signaling has been linked with a host of cardiovascular phenotypes including heart failure and arrhythmia, and CaMKII levels are elevated in human and animal disease models of heart disease. While nearly a decade has been invested in targeting CaMKII for the treatment of heart failure and arrhythmia phenotypes, to date, approaches to target the molecule for antiarrhythmic benefit have been unsuccessful for reasons that are still not entirely clear, although (1) lack of compound specificity and (2) the multitude of downstream targets are likely contributing factors. This review will provide an update on current pathways regulated by CaMKII with the goal of illustrating potential upstream regulatory mechanisms and downstream targets that may be modulated for the prevention of cardiac electrical defects. While the review will cover multiple aspects of CaMKII dysfunction in cardiovascular disease, we have given special attention to the potential of CaMKII-associated late Na+ current as a novel therapeutic target for cardiac arrhythmia.
Computational biology is a powerful tool for elucidating arrhythmogenic mechanisms at the cellular level, where complex interactions between ionic processes determine behavior. A novel theoretical ...model of the canine ventricular epicardial action potential and calcium cycling was developed and used to investigate ionic mechanisms underlying Ca2+ transient (CaT) and action potential duration (APD) rate dependence.
The Ca2+/calmodulin-dependent protein kinase (CaMKII) regulatory pathway was integrated into the model, which included a novel Ca2+-release formulation, Ca2+ subspace, dynamic chloride handling, and formulations for major ion currents based on canine ventricular data. Decreasing pacing cycle length from 8000 to 300 ms shortened APD primarily because of I(Ca(L)) reduction, with additional contributions from I(to1), I(NaK), and late I(Na). CaT amplitude increased as cycle length decreased from 8000 to 500 ms. This positive rate-dependent property depended on CaMKII activity.
CaMKII is an important determinant of the rate dependence of CaT but not of APD, which depends on ion-channel kinetics. The model of CaMKII regulation may serve as a paradigm for modeling effects of other regulatory pathways on cell function.
BACKGROUND—Voltage-gated Na channels (Nav) are essential for myocyte membrane excitability and cardiac function. Nav current (INa) is a large-amplitude, short-duration spike generated by rapid ...channel activation followed immediately by inactivation. However, even under normal conditions, a small late component of INa (INa,L) persists because of incomplete/failed inactivation of a subpopulation of channels. Notably, INa,L is directly linked with both congenital and acquired disease states. The multifunctional Ca/calmodulin-dependent kinase II (CaMKII) has been identified as an important activator of INa,L in disease. Several potential CaMKII phosphorylation sites have been discovered, including Ser571 in the Nav1.5 DI-DII linker, but the molecular mechanism underlying CaMKII-dependent regulation of INa,L in vivo remains unknown.
METHODS AND RESULTS—To determine the in vivo role of Ser571, 2 Scn5a knock-in mouse models were generated expressing either(1) Nav1.5 with a phosphomimetic mutation at Ser571 (S571E), or (2) Nav1.5 with the phosphorylation site ablated (S571A). Electrophysiology studies revealed that Ser571 regulates INa,L but not other channel properties previously linked to CaMKII. Ser571-mediated increases in INa,L promote abnormal repolarization and intracellular Ca handling and increase susceptibility to arrhythmia at the cellular and animal level. Importantly, Ser571 is required for maladaptive remodeling and arrhythmias in response to pressure overload.
CONCLUSIONS—Our data provide the first in vivo evidence for the molecular mechanism underlying CaMKII activation of the pathogenic INa,L. Relevant for improved rational design of potential therapies, our findings demonstrate that Ser571-dependent regulation of Nav1.5 specifically tunes INa,L without altering critical physiological components of the current.
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