Conserved Role of the Large Conductance Calcium-Activated Potassium Channel, KCa1.1, in Sinus Node Function and Arrhythmia Risk.

BACKGROUND: KCNMA1 encodes the α-subunit of the large-conductance Ca2+-activated K+ channel, KCa1.1, and lies within a linkage interval for atrial fibrillation (AF). Insights into the cardiac functions of KCa1.1 are limited, and KCNMA1 has not been investigated as an AF candidate gene. METHODS: The KCNMA1 gene was sequenced in 118 patients with familial AF. The role of KCa1.1 in normal cardiac structure and function was evaluated in humans, mice, zebrafish, and fly. A novel KCNMA1 variant was functionally characterized. RESULTS: A complex KCNMA1 variant was identified in 1 kindred with AF. To evaluate potential disease mechanisms, we first evaluated the distribution of KCa1.1 in normal hearts using immunostaining and immunogold electron microscopy. KCa1.1 was seen throughout the atria and ventricles in humans and mice, with strong expression in the sinus node. In an ex vivo murine sinoatrial node preparation, addition of the KCa1.1 antagonist, paxilline, blunted the increase in beating rate induced by adrenergic receptor stimulation. Knockdown of the KCa1.1 ortholog, kcnma1b, in zebrafish embryos resulted in sinus bradycardia with dilatation and reduced contraction of the atrium and ventricle. Genetic inactivation of the Drosophila KCa1.1 ortholog, slo, systemically or in adult stages, also slowed the heartbeat and produced fibrillatory cardiac contractions. Electrophysiological characterization of slo-deficient flies revealed bursts of action potentials, reflecting increased events of fibrillatory arrhythmias. Flies with cardiac-specific overexpression of the human KCNMA1 mutant also showed increased heart period and bursts of action potentials, similar to the KCa1.1 loss-of-function models. CONCLUSIONS: Our data point to a highly conserved role of KCa1.1 in sinus node function in humans, mice, zebrafish, and fly and suggest that KCa1.1 loss of function may predispose to AF.

Novel genomic targets of valosin-containing protein in protecting pathological cardiac hypertrophy.

Pressure overload-induced cardiac hypertrophy, such as that caused by hypertension, is a key risk factor for heart failure. However, the underlying molecular mechanisms remain largely unknown. We previously reported that the valosin-containing protein (VCP), an ATPase-associated protein newly identified in the heart, acts as a significant mediator of cardiac protection against pressure overload-induced pathological cardiac hypertrophy. Still, the underlying molecular basis for the protection is unclear. This study used a cardiac-specific VCP transgenic mouse model to understand the transcriptomic alterations induced by VCP under the cardiac stress caused by pressure overload. Using RNA sequencing and comprehensive bioinformatic analysis, we found that overexpression of the VCP in the heart was able to normalize the pressure overload-stimulated hypertrophic signals by activating G protein-coupled receptors, particularly, the olfactory receptor family, and inhibiting the transcription factor controlling cell proliferation and differentiation. Moreover, VCP overexpression restored pro-survival signaling through regulating alternative splicing alterations of mitochondrial genes. Together, our study revealed a novel molecular regulation mediated by VCP under pressure overload that may bring new insight into the mechanisms involved in protecting against hypertensive heart failure.

Genomic characterization reveals novel mechanisms underlying the valosin-containing protein-mediated cardiac protection against heart failure.

Chronic hypertension is a key risk factor for heart failure. However, the underlying molecular mechanisms are not fully understood. Our previous studies found that the valosin-containing protein (VCP), an ATPase-associated protein, was significantly decreased in the hypertensive heart tissues. In this study, we tested the hypothesis that restoration of VCP protected the heart against pressure overload-induced heart failure. With a cardiac-specific transgenic (TG) mouse model, we showed that a moderate increase of VCP was able to attenuate chronic pressure overload-induced maladaptive cardiac hypertrophy and dysfunction. RNA sequencing and a comprehensive bioinformatic analysis further demonstrated that overexpression of VCP in the heart normalized the pressure overload-stimulated hypertrophic signals and repressed the stress-induced inflammatory response. In addition, VCP overexpression promoted cell survival by enhancing the mitochondria resistance to the oxidative stress via activating the Rictor-mediated-gene networks. VCP was also found to be involved in the regulation of the alternative splicing and differential isoform expression for some genes that are related to ATP production and protein synthesis by interacting with long no-coding RNAs and histone deacetylases, indicating a novel epigenetic regulation of VCP in integrating coding and noncoding genomic network in the stressed heart. In summary, our study demonstrated that the rescuing of a deficient VCP in the heart could prevent pressure overload-induced heart failure by rectifying cardiac hypertrophic and inflammatory signaling and enhancing the cardiac resistance to oxidative stress, which brought in novel insights into the understanding of the mechanism of VCP in protecting patients from hypertensive heart failure.

The Valosin-Containing Protein Protects the Heart Against Pathological Ca2+ Overload by Modulating Ca2+ Uptake Proteins.

Stress-induced mitochondrial calcium (Ca2+) overload is a key cellular toxic effectors and a trigger of cardiomyocyte death during cardiac ischemic injury through the opening of mitochondrial permeability transition pore (mPTP). We previously found that the valosin-containing protein (VCP), an ATPase-associated protein, protects cardiomyocytes against stress-induced death and also inhibits mPTP opening in vitro. However, the underlying molecular mechanisms are not fully understood. Here, we tested our hypothesis that VCP acts as a novel regulator of mitochondrial Ca2+ uptake proteins and resists cardiac mitochondrial Ca2+ overload by modulating mitochondrial Ca2+ homeostasis. By using a cardiac-specific transgenic (TG) mouse model in which VCP is overexpressed by 3.5 folds in the heart compared to the wild type (WT) mouse, we found that, under the pathological extra-mitochondrial Ca2+ overload, Ca2+ entry into cardiac mitochondria was reduced in VCP TG mice compared to their little-matched WT mice, subsequently preventing mPTP opening and ATP depletion under the Ca2+ challenge. Mechanistically, overexpression of VCP in the heart resulted in post-translational protein degradation of the mitochondrial Ca2+ uptake protein 1, an activator of the mitochondria Ca2+ uniporter that is responsible for mitochondrial calcium uptake. Together, our results reveal a new regulatory role of VCP in cardiac mitochondrial Ca2+ homeostasis and unlock the potential mechanism by which VCP confers its cardioprotection.

Non-Coding RNA in the Pathogenesis, Progression and Treatment of Hypertension.

Hypertension is a complex, multifactorial disease that involves the coexistence of multiple risk factors, environmental factors and physiological systems. The complexities extend to the treatment and management of hypertension, which are still the pursuit of many researchers. In the last two decades, various genes have emerged as possible biomarkers and have become the target for investigations of specialized drug design based on its risk factors and the primary cause. Owing to the growing technology of microarrays and next-generation sequencing, the non-protein-coding RNAs (ncRNAs) have increasingly gained attention, and their status of redundancy has flipped to importance in normal cellular processes, as well as in disease progression. The ncRNA molecules make up a significant portion of the human genome, and their role in diseases continues to be uncovered. Specifically, the cellular role of these ncRNAs has played a part in the pathogenesis of hypertension and its progression to heart failure. This review explores the function of the ncRNAs, their types and biology, the current update of their association with hypertension pathology and the potential new therapeutic regime for hypertension.

Nesprin-1 and actin contribute to nuclear and cytoskeletal defects in lamin A/C-deficient cardiomyopathy.

Lamin A/C mutations are the most common cause of familial dilated cardiomyopathy (DCM) but the pathogenetic mechanisms are incompletely understood. Nesprins are spectrin repeat-containing proteins that interact with lamin A/C and are components of the linker-of-nucleoskeleton-and-cytoskeleton (LINC) complex that connects the nuclear envelope to the actin cytoskeleton. Our aim was to determine whether changes in nesprin-1 and actin might contribute to DCM in homozygous Lmna knockout (Lmna(-/-)) mice. Here we find that Lmna(-/-) cardiomyocytes have altered nuclear envelope morphology, disorganization of nesprin-1 and heterogeneity in the distribution of nuclear and cytoskeletal actin. Functional interactions of nesprin-1 with nuclear G-actin and with the cytoskeletal γ-actin, α-cardiac actin and α-smooth muscle actin (α-SMA) isoforms were shown by immunoprecipitation and Western blotting. At 4-6 weeks of age, Lmna(-/-) mice had normal levels of γ-actin and α-cardiac actin, but α-SMA expression was increased by 50%. In contrast to the predominant vascular distribution of α-SMA in WT ventricular sections, α-SMA had a diffuse staining pattern in Lmna(-/-) sections. Osmotic swelling studies showed enhanced radial swelling in Lmna(-/-) cardiomyocytes indicative of cytoskeletal instability. The distensibility of Lmna(-/-) cardiomyocytes with osmotic stress was reduced by addition of α-SMA-specific fusion peptide. Our findings support a model in which uncoupling of the nucleus and cytoskeleton associated with disruption of the LINC complex promotes mechanical instability and defective force transmission in cardiomyocytes. Changes in the distribution and expression patterns of nuclear and cytoskeletal actin suggest that diverse transcriptional and structural defects may also contribute to DCM in Lmna(-/-) mice.

Effects of mechanical stress and carvedilol in lamin A/C-deficient dilated cardiomyopathy.

RATIONALE: Mutations in the LMNA gene, which encodes the nuclear lamina proteins lamin A and lamin C, are the most common cause of familial dilated cardiomyopathy (DCM). Mechanical stress-induced apoptosis has been proposed as the mechanism underpinning DCM in lamin A/C-deficient hearts, but supporting in vivo evidence has been lacking. OBJECTIVE: Our aim was to study interventions to modify mechanical stress in heterozygous Lmna knockout (Lmna(+/-)) mice. METHODS AND RESULTS: Cardiac structure and function were evaluated before and after exercise training, thoracic aortic constriction, and carvedilol treatment. Lmna(+/-) mice develop adult-onset DCM with relatively more severe disease in males. Lmna(+/-) cardiomyocytes show altered nuclear morphology and perinuclear desmin organization, with enhanced responses to hypo-osmotic stress indicative of cytoskeletal instability. Despite these structural defects that provide a template for mechanical stress-induced damage, young Lmna(+/-) mice subjected to 6 weeks of moderate or strenuous exercise training did not show induction of apoptosis or accelerated DCM. In contrast, regular moderate exercise attenuated DCM development in male Lmna(+/-) mice. Sustained pressure overload generated by thoracic aortic constriction depressed ventricular contraction in young wild-type and Lmna(+/-) mice with no sex or genotype differences in the time-course or severity of response. Treatment of male Lmna(+/-) mice from 12 to 40 weeks with the beta-blocker, carvedilol, prevented the dilatation and contractile dysfunction that was observed in placebo-treated mice. CONCLUSIONS: These data suggest that factors other than mechanical stress-induced apoptosis contribute to DCM and provide the first demonstration that regular moderate exercise and carvedilol can modify disease progression in lamin A/C-deficient hearts.

Murine T-box transcription factor Tbx20 acts as a repressor during heart development, and is essential for adult heart integrity, function and adaptation.

The genetic hierarchies guiding lineage specification and morphogenesis of the mammalian embryonic heart are poorly understood. We now show by gene targeting that murine T-box transcription factor Tbx20 plays a central role in these pathways, and has important activities in both cardiac development and adult function. Loss of Tbx20 results in death of embryos at mid-gestation with grossly abnormal heart morphogenesis. Underlying these disturbances was a severely compromised cardiac transcriptional program, defects in the molecular pre-pattern, reduced expansion of cardiac progenitors and a block to chamber differentiation. Notably, Tbx20-null embryos showed ectopic activation of Tbx2 across the whole heart myogenic field. Tbx2 encodes a transcriptional repressor normally expressed in non-chamber myocardium, and in the atrioventricular canal it has been proposed to inhibit chamber-specific gene expression through competition with positive factor Tbx5. Our data demonstrate a repressive activity for Tbx20 and place it upstream of Tbx2 in the cardiac genetic program. Thus, hierarchical, repressive interactions between Tbx20 and other T-box genes and factors underlie the primary lineage split into chamber and non-chamber myocardium in the forming heart, an early event upon which all subsequent morphogenesis depends. Additional roles for Tbx20 in adult heart integrity and contractile function were revealed by in-vivo cardiac functional analysis of Tbx20 heterozygous mutant mice. These data suggest that mutations in human cardiac transcription factor genes, possibly including TBX20, underlie both congenital heart disease and adult cardiomyopathies.

Defects in nuclear structure and function promote dilated cardiomyopathy in lamin A/C-deficient mice.

Laminopathies are a group of disorders caused by mutations in the LMNA gene that encodes the nuclear lamina proteins, lamin A and lamin C; their pathophysiological basis is unknown. We report that lamin A/C-deficient (Lmna(-/-)) mice develop rapidly progressive dilated cardiomyopathy (DCM) characterized by left ventricular (LV) dilation and reduced systolic contraction. Isolated Lmna(-/-) myocytes show reduced shortening with normal baseline and peak amplitude of Ca(2+) transients. Lmna(-/-) LV myocyte nuclei have marked alterations of shape and size with central displacement and fragmentation of heterochromatin; these changes are present but less severe in left atrial nuclei. Electron microscopy of Lmna(-/-) cardiomyocytes shows disorganization and detachment of desmin filaments from the nuclear surface with progressive disruption of the cytoskeletal desmin network. Alterations in nuclear architecture are associated with defective nuclear function evidenced by decreased SREBP1 import, reduced PPARgamma expression, and a lack of hypertrophic gene activation. These findings suggest a model in which the primary pathophysiological mechanism in Lmna(-/-) mice is defective force transmission resulting from disruption of lamin interactions with the muscle-specific desmin network and loss of cytoskeletal tension. Despite severe DCM, defects in nuclear function prevent Lmna(-/-) cardiomyocytes from developing compensatory hypertrophy and accelerate disease progression.