Key points
Association of plasma membrane BKCa channels with BK‐β subunits shapes their biophysical properties and physiological roles; however, functional modulation of the mitochondrial BKCa ...channel (mitoBKCa) by BK‐β subunits is not established.
MitoBKCa‐α and the regulatory BK‐β1 subunit associate in mouse cardiac mitochondria.
A large fraction of mitoBKCa display properties similar to that of plasma membrane BKCa when associated with BK‐β1 (left‐shifted voltage dependence of activation, V1/2 = −55 mV, 12 µm matrix Ca2+).
In BK‐β1 knockout mice, cardiac mitoBKCa displayed a low Po and a depolarized V1/2 of activation (+47 mV at 12 µm matrix Ca2+)
Co‐expression of BKCa with the BK‐β1 subunit in HeLa cells doubled the density of BKCa in mitochondria.
The present study supports the view that the cardiac mitoBKCa channel is functionally modulated by the BK‐β1 subunit; proper targeting and activation of mitoBKCa shapes mitochondrial Ca2+ handling.
Association of the plasma membrane BKCa channel with auxiliary BK‐β1–4 subunits profoundly affects the regulatory mechanisms and physiological processes in which this channel participates. However, functional association of mitochondrial BK (mitoBKCa) with regulatory subunits is unknown. We report that mitoBKCa functionally associates with its regulatory subunit BK‐β1 in adult rodent cardiomyocytes. Cardiac mitoBKCa is a calcium‐ and voltage‐activated channel that is sensitive to paxilline with a large conductance for K+ of 300 pS. Additionally, mitoBKCa displays a high open probability (Po) and voltage half‐activation (V1/2 = −55 mV, n = 7) resembling that of plasma membrane BKCa when associated with its regulatory BK‐β1 subunit. Immunochemistry assays demonstrated an interaction between mitochondrial BKCa‐α and its BK‐β1 subunit. Mitochondria from the BK‐β1 knockout (KO) mice showed sparse mitoBKCa currents (five patches with mitoBKCa activity out of 28 total patches from n = 5 different hearts), displaying a depolarized V1/2 of activation (+47 mV in 12 µm matrix Ca2+). The reduced activity of mitoBKCa was accompanied by a high expression of BKCa transcript in the BK‐β1 KO, suggesting a lower abundance of mitoBKCa channels in this genotype. Accordingly, BK‐β1subunit increased the localization of BKDEC (i.e. the splice variant of BKCa that specifically targets mitochondria) into mitochondria by two‐fold. Importantly, both paxilline‐treated and BK‐β1 KO mitochondria displayed a more rapid Ca2+ overload, featuring an early opening of the mitochondrial transition pore. We provide strong evidence that mitoBKCa associates with its regulatory BK‐β1 subunit in cardiac mitochondria, ensuring proper targeting and activation of the mitoBKCa channel that helps to maintain mitochondrial Ca2+ homeostasis.
Key points
Association of plasma membrane BKCa channels with BK‐β subunits shapes their biophysical properties and physiological roles; however, functional modulation of the mitochondrial BKCa channel (mitoBKCa) by BK‐β subunits is not established.
MitoBKCa‐α and the regulatory BK‐β1 subunit associate in mouse cardiac mitochondria.
A large fraction of mitoBKCa display properties similar to that of plasma membrane BKCa when associated with BK‐β1 (left‐shifted voltage dependence of activation, V1/2 = −55 mV, 12 µm matrix Ca2+).
In BK‐β1 knockout mice, cardiac mitoBKCa displayed a low Po and a depolarized V1/2 of activation (+47 mV at 12 µm matrix Ca2+)
Co‐expression of BKCa with the BK‐β1 subunit in HeLa cells doubled the density of BKCa in mitochondria.
The present study supports the view that the cardiac mitoBKCa channel is functionally modulated by the BK‐β1 subunit; proper targeting and activation of mitoBKCa shapes mitochondrial Ca2+ handling.
Epigenomes in Cardiovascular Disease Rosa-Garrido, Manuel; Chapski, Douglas J; Vondriska, Thomas M
Circulation research,
2018-May-25, Letnik:
122, Številka:
11
Journal Article
Recenzirano
Odprti dostop
If unifying principles could be revealed for how the same genome encodes different eukaryotic cells and for how genetic variability and environmental input are integrated to impact cardiovascular ...health, grand challenges in basic cell biology and translational medicine may succumb to experimental dissection. A rich body of work in model systems has implicated chromatin-modifying enzymes, DNA methylation, noncoding RNAs, and other transcriptome-shaping factors in adult health and in the development, progression, and mitigation of cardiovascular disease. Meanwhile, deployment of epigenomic tools, powered by next-generation sequencing technologies in cardiovascular models and human populations, has enabled description of epigenomic landscapes underpinning cellular function in the cardiovascular system. This essay aims to unpack the conceptual framework in which epigenomes are studied and to stimulate discussion on how principles of chromatin function may inform investigations of cardiovascular disease and the development of new therapies.
Endothelial cells contribute to a subset of cardiac fibroblasts by undergoing endothelial-to-mesenchymal transition, but whether cardiac fibroblasts can adopt an endothelial cell fate and directly ...contribute to neovascularization after cardiac injury is not known. Here, using genetic fate map techniques, we demonstrate that cardiac fibroblasts rapidly adopt an endothelial-cell-like phenotype after acute ischaemic cardiac injury. Fibroblast-derived endothelial cells exhibit anatomical and functional characteristics of native endothelial cells. We show that the transcription factor p53 regulates such a switch in cardiac fibroblast fate. Loss of p53 in cardiac fibroblasts severely decreases the formation of fibroblast-derived endothelial cells, reduces post-infarct vascular density and worsens cardiac function. Conversely, stimulation of the p53 pathway in cardiac fibroblasts augments mesenchymal-to-endothelial transition, enhances vascularity and improves cardiac function. These observations demonstrate that mesenchymal-to-endothelial transition contributes to neovascularization of the injured heart and represents a potential therapeutic target for enhancing cardiac repair.
Modulating the number of muscle stems cells, called satellite cells, during early postnatal development produces long‐term effects on muscle growth. We tested the hypothesis that high expression ...levels of the anti‐aging protein Klotho in early postnatal myogenesis increase satellite cell numbers by influencing the epigenetic regulation of genes that regulate myogenesis. Our findings show that elevated klotho expression caused a transient increase in satellite cell numbers and slowed muscle fiber growth, followed by a period of accelerated muscle growth that leads to larger fibers. Klotho also transcriptionally downregulated the H3K27 demethylase Jmjd3, leading to increased H3K27 methylation and decreased expression of genes in the canonical Wnt pathway, which was associated with a delay in muscle differentiation. In addition, Klotho stimulation and Jmjd3 downregulation produced similar but not additive reductions in the expression of Wnt4, Wnt9a, and Wnt10a in myogenic cells, indicating that inhibition occurred through a common pathway. Together, our results identify a novel pathway through which Klotho influences myogenesis by reducing the expression of Jmjd3, leading to reductions in the expression of Wnt genes and inhibition of canonical Wnt signaling.
Association of plasma membrane BK
channels with BK-β subunits shapes their biophysical properties and physiological roles; however, functional modulation of the mitochondrial BK
channel (mitoBK
) by ...BK-β subunits is not established. MitoBK
-α and the regulatory BK-β1 subunit associate in mouse cardiac mitochondria. A large fraction of mitoBK
display properties similar to that of plasma membrane BK
when associated with BK-β1 (left-shifted voltage dependence of activation, V
= -55 mV, 12 µm matrix Ca
). In BK-β1 knockout mice, cardiac mitoBK
displayed a low P
and a depolarized V
of activation (+47 mV at 12 µm matrix Ca
) Co-expression of BK
with the BK-β1 subunit in HeLa cells doubled the density of BK
in mitochondria. The present study supports the view that the cardiac mitoBK
channel is functionally modulated by the BK-β1 subunit; proper targeting and activation of mitoBK
shapes mitochondrial Ca
handling.
Association of the plasma membrane BK
channel with auxiliary BK-β1-4 subunits profoundly affects the regulatory mechanisms and physiological processes in which this channel participates. However, functional association of mitochondrial BK (mitoBK
) with regulatory subunits is unknown. We report that mitoBK
functionally associates with its regulatory subunit BK-β1 in adult rodent cardiomyocytes. Cardiac mitoBK
is a calcium- and voltage-activated channel that is sensitive to paxilline with a large conductance for K
of 300 pS. Additionally, mitoBK
displays a high open probability (P
) and voltage half-activation (V
= -55 mV, n = 7) resembling that of plasma membrane BK
when associated with its regulatory BK-β1 subunit. Immunochemistry assays demonstrated an interaction between mitochondrial BK
-α and its BK-β1 subunit. Mitochondria from the BK-β1 knockout (KO) mice showed sparse mitoBK
currents (five patches with mitoBK
activity out of 28 total patches from n = 5 different hearts), displaying a depolarized V
of activation (+47 mV in 12 µm matrix Ca
). The reduced activity of mitoBK
was accompanied by a high expression of BK
transcript in the BK-β1 KO, suggesting a lower abundance of mitoBK
channels in this genotype. Accordingly, BK-β1subunit increased the localization of BKDEC (i.e. the splice variant of BK
that specifically targets mitochondria) into mitochondria by two-fold. Importantly, both paxilline-treated and BK-β1 KO mitochondria displayed a more rapid Ca
overload, featuring an early opening of the mitochondrial transition pore. We provide strong evidence that mitoBK
associates with its regulatory BK-β1 subunit in cardiac mitochondria, ensuring proper targeting and activation of the mitoBK
channel that helps to maintain mitochondrial Ca
homeostasis.
Abstract
Heart failure with preserved ejection fraction (HFpEF) exhibits a sex bias, being more common in women than men, and we hypothesize that mitochondrial sex differences might underlie this ...bias. As part of genetic studies of heart failure in mice, we observe that heart mitochondrial DNA levels and function tend to be reduced in females as compared to males. We also observe that expression of genes encoding mitochondrial proteins are higher in males than females in human cohorts. We test our hypothesis in a panel of genetically diverse inbred strains of mice, termed the Hybrid Mouse Diversity Panel (HMDP). Indeed, we find that mitochondrial gene expression is highly correlated with diastolic function, a key trait in HFpEF. Consistent with this, studies of a “two-hit” mouse model of HFpEF confirm that mitochondrial function differs between sexes and is strongly associated with a number of HFpEF traits. By integrating data from human heart failure and the mouse HMDP cohort, we identify the mitochondrial gene
Acsl6
as a genetic determinant of diastolic function. We validate its role in HFpEF using adenoviral over-expression in the heart. We conclude that sex differences in mitochondrial function underlie, in part, the sex bias in diastolic function.
The mutagenicy of the slightly weathered fuel oil from the Prestige oil spill and the effects of different bioremediation products (nutrients and/or microorganisms and biodiesel) on the potential ...mutagenic activity of this heavy fuel oil spilled on the shore were evaluated for a period of 1
year using the Ames
Salmonella assay with strains TA98, TA100, TA1535 and TA1537 in the absence and presence of exogenous metabolic activation (S9 fraction from rat liver). The in situ bioremediation experiment was performed using tiles located in the supra-littoral and intertidal zones of a beach seriously affected by the fuel oil spill. The results obtained showed the mutagenic activity of the slightly weathered fuel oil extracts at the beginning of the experiment in strain TA98 that persisted for more than 150
days in both the untreated control and treated tiles independently of the zone of the beach considered. However, after 360
days neither the control nor the treated tiles in the intertidal zone showed mutagenic activity and a weak positive response in strain TA98 was detected for the control fuel oil extracts from supra-littoral tiles. The application of biodiesel to accelerate the biodegradation of this type of fuel oil may constitute a further genotoxic hazard to the environment, since the mutagenic response achieved from the biodiesel–fuel oil mixture in the first samplings (days 0 and 30) was more potent than that obtained from the control tiles. The mutagenic activity was detected along the study with
S. typhimurium TA98 in both the presence and absence of S9 microsomal fraction, but the addition of S9 fraction in the assay always increased the number of revertants induced. In general, these findings suggest that the bioremediation strategies used were not effective in eliminating the genotoxic hazard associated with this heavy fuel oil attached to rocky substrate since they did not achieve a decrease in the mutagenic response with respect to the untreated control tiles. These data also confirm that genotoxicity assays should be used to evaluate the effectiveness of bioremediation efforts associated with oil spills for a better risk assessment.
► The mutagenic activity of the slightly weathered fuel oil from the Prestige oil spill persisted for more than 150
days. ► The bioremediation strategies used were not effective in eliminating the genotoxic hazard. ► The application of biodiesel may constitute a further genotoxic hazard to the environment.
Recent technological advancements in the field of chromatin biology have rewritten the textbook on nuclear organization. We now appreciate that the folding of chromatin in the three-dimensional space ...(i.e. its 3D “architecture”) is non-random, hierarchical, and highly complex. While 3D chromatin structure is partially encoded in the primary sequence and thereby broadly conserved across cell types and states, a substantial portion of the genome seems to be dynamic during development or in disease. Moreover, there is growing evidence that at least some of the 3D structure of chromatin is functionally linked to gene regulation, both being modulated by and impacting on multiple nuclear processes (including DNA replication, transcription, and RNA splicing). In recent years, these new concepts have nourished several investigations about the functional role of 3D chromatin topology dynamics in the heart during development and disease. This review aims to provide a comprehensive overview of our current understanding in this field, and to discuss how this knowledge can inform further research as well as clinical practice.
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•Chromatin folding in three dimensions is non-random, hierarchical, and functional.•Cardiomyocyte differentiation changes intra- and inter-chromosomal interactions.•Mutations in nuclear factors can disrupt developmental chromatin folding dynamics.•Heart disease-associated genetic variants are often involved in chromatin looping.•Environmental stress can alter 3D chromatin organization in the adult heart.
•The study checked the level of achievement of defined quality standards for outcomes.•The Spanish emergency departments (ED) are quite far from fulfilling these proposed standards.•Experts ...recommended direct discharge for EDs able to observe acute heart failure patients should be over 40%.•Only 12% of Spanish EDs achieve the 40% of direct discharge.•High percentages of direct discharge from ED negatively impact revisit or hospitalization but not mortality.
Experts recommended that direct discharge without hospitalization (DDWH) for emergency departments (EDs) able to observe acute heart failure (AHF) patients should be >40%, and these discharged patients should fulfil the following outcome standards: 30-day all-cause mortality <2% (outcome A); 7-day ED revisit due to AHF < 10% (outcome B); and 30-day ED revisit/hospitalization due to AHF < 20% (outcome C). We investigated these outcomes in a nationwide cohort and their relationship with the ED DDWH percentage.
We analyzed the EAHFE registry (includes about 15% of Spanish EDs), calculated DDWH percentage of each ED, and A/B/C outcomes of DDWH patients, overall and in each individual ED. Relationship between ED DDWH and outcomes was assessed by linear and quadratic regression models, non-weighted and weighted by DDWH patients provided by each ED.
Among 17,420 patients, 4488 had DDWH (25.8%, median ED stay = 0 days, IQR = 0–1). Only 12.9% EDs achieved DDWH > 40%. Considering DDWH patients altogether, outcomes A/C were above the recommended standards (4.3%/29.4%), while outcome B was nearly met (B = 10.1%). When analyzing individual EDs, 58.1% of them achieved the outcome B standard, while outcomes A/C standards were barely achieved (19.3%/9.7%). We observed clinically relevant linear/quadratic relationships between higher DDWH and worse outcomes B (weighted R2 = 0.184/0.322) and C (weighted R2 = 0.430/0.624), but not with outcome A (weighted R2 = 0.002/0.022).
The EDs of this nationwide cohort do not fulfil the standards for AHF patients with DDWH. High DDWH rates negatively impact ED revisit or hospitalization but not mortality. This may represent an opportunity for improvement in better selecting patients for early ED discharge and in ensuring early follow-up after ED discharge.
Abstract only Heart failure is a critical health issue that causes one in nine deaths in the United States. Despite extensive research on gene expression dynamics in the failing myocardium, the ...mechanisms by which the genome reorganizes itself to reconfigure transcription are poorly understood. CTCF is a crucial protein in chromatin architecture that mediates chromatin looping, acts as an enhancer insulator, and can function as either an activator or repressor of transcription. Our previous research indicates that CTCF plays a crucial role in heart failure, as cardiac-specific knockout of this protein promotes genome organization defects that result in cardiac pathology marked by reduced ejection fraction. To determine whether restoring CTCF levels could reverse this phenotype, we designed an AAV9 vector in which the cardiomyocyte-specific Troponin T promoter drives the expression of CTCF and GFP. Our results show that reintroduction of CTCF into failing CTCF-depleted mouse hearts successfully rescues the pathological phenotype of the animals, characterized by sustained improvement in ejection fraction and left ventricular chamber size in both systole and diastole. Furthermore, RNA-seq experiments reveal numerous differentially expressed genes with opposite transcriptional behavior depending on CTCF levels, including genes involved in chromatin organization (e.g., CHmgb2, Brca1, Smyd2), development of hypertrophy (e.g., Mef2a, Gata4, Mybpc3), extracellular matrix organization (e.g., Adamts5, Itgb1, Vtn) and collagen deposition (e.g., Col5a1, Col5a3). We also examined chromatin architecture through Hi-C experiments and discovered that restoration of CTCF levels remodels the global architecture of chromatin to closely resemble healthy myocyte chromatin. This finding supports the hypothesis that rescue of CTCF levels ameliorates cardiac disease by reverting the observed pathological gene expression profile through genomic reorganization. In summary, our project highlights the crucial role of CTCF in heart failure and demonstrates that structural repair of chromatin organization through modulation of CTCF levels is a novel and realistic approach to treating heart failure.
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