Novel cholesterol-dependent regulation of cardiac KATP subunit expression revealed using histone deacetylase inhibitors.

We recently discovered that the histone deacetylase inhibitor, trichostatin A (TSA), increases expression of the sulfonylurea receptor 2 (SUR2; Abcc9) subunit of the ATP-sensitive K+ (KATP ) channel in HL-1 cardiomyocytes. Interestingly, the increase in SUR2 was abolished with exogenous cholesterol, suggesting that cholesterol may regulate channel expression. In the present study, we tested the hypothesis that TSA increases SUR2 by depleting cholesterol and activating the sterol response element binding protein (SREBP) family of transcription factors. Treatment of HL-1 cardiomyocytes with TSA (30 ng/ml) caused a time-dependent increase in SUR2 mRNA expression that correlates with the time course of cholesterol depletion assessed by filipin staining. Consistent with the cholesterol-dependent regulation of SREBP increasing SUR2 mRNA expression, we observe a significant increase in SREBP cleavage and translocation to the nucleus following TSA treatment that is inhibited by exogenous cholesterol. Further supporting the role of SREBP in mediating the effect of TSA on KATP subunit expression, SREBP1 significantly increased luciferase reporter gene expression driven by the upstream SUR2 promoter. Lastly, HL-1 cardiomyocytes treated with the SREBP inhibitor PF429242 significantly suppresses the effect of TSA on SUR2 gene expression. These results demonstrate that SREBP is an important regulator of KATP channel expression and suggest a novel method by which hypercholesterolemia may exert negative effects on the cardiovascular system, namely, by suppressing expression of the KATP channel.

SMN-deficiency disrupts SERCA2 expression and intracellular Ca2+ signaling in cardiomyocytes from SMA mice and patient-derived iPSCs.

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by loss of alpha motor neurons and skeletal muscle atrophy. The disease is caused by mutations of the SMN1 gene that result in reduced functional expression of survival motor neuron (SMN) protein. SMN is ubiquitously expressed, and there have been reports of cardiovascular dysfunction in the most severe SMA patients and animal models of the disease. In this study, we directly assessed the function of cardiomyocytes isolated from a severe SMA model mouse and cardiomyocytes generated from patient-derived IPSCs. Consistent with impaired cardiovascular function at the very early disease stages in mice, heart failure markers such as brain natriuretic peptide were significantly elevated. Functionally, cardiomyocyte relaxation kinetics were markedly slowed and the T50 for Ca2+ sequestration increased to 146 ± 4 ms in SMN-deficient cardiomyocytes from 126 ± 4 ms in wild type cells. Reducing SMN levels in cardiomyocytes from control patient IPSCs slowed calcium reuptake similar to SMA patent-derived cardiac cells. Importantly, restoring SMN increased calcium reuptake rate. Taken together, these results indicate that SMN deficiency impairs cardiomyocyte function at least partially through intracellular Ca2+ cycling dysregulation.

Histone deacetylase inhibitors modulate KATP subunit transcription in HL-1 cardiomyocytes through effects on cholesterol homeostasis.

Histone deacetylase inhibitors (HDIs) are under investigation for the treatment of a number of human health problems. HDIs have proven therapeutic value in refractory cases of cutaneous T-cell lymphoma. Electrocardiographic ST segment morphological changes associated with HDIs were observed during development. Because ST segment morphology is typically linked to changes in ATP sensitive potassium (KATP) channel activity, we tested the hypothesis that HDIs affect cardiac KATP channel subunit expression. Two different HDIs, romidepsin and trichostatin A, caused ~20-fold increase in SUR2 (Abcc9) subunit mRNA expression in HL-1 cardiomyocytes. The effect was specific for the SUR2 subunit as neither compound causes a marked change in SUR1 (Abcc8) expression. Moreover, the effect was cell specific as neither HDI markedly altered KATP subunit expression in MIN6 pancreatic ß-cells. We observe significant enrichment of the H3K9Ac histone mark specifically at the SUR2 promoter consistent with the conclusion that chromatin remodeling at this locus plays a role in increasing SUR2 gene expression. Unexpectedly, however, we also discovered that HDI-dependent depletion of cellular cholesterol is required for the observed effects on SUR2 expression. Taken together, the data in the present study demonstrate that KATP subunit expression can be epigenetically regulated in cardiomyocytes, defines a role for cholesterol homeostasis in mediating epigenetic regulation and suggests a potential molecular basis for the cardiac effects of the HDIs.

Acetate transiently inhibits myocardial contraction by increasing mitochondrial calcium uptake.

BACKGROUND: There is a close relationship between cardiovascular disease and cardiac energy metabolism, and we have previously demonstrated that palmitate inhibits myocyte contraction by increasing Kv channel activity and decreasing the action potential duration. Glucose and long chain fatty acids are the major fuel sources supporting cardiac function; however, cardiac myocytes can utilize a variety of substrates for energy generation, and previous studies demonstrate the acetate is rapidly taken up and oxidized by the heart. In this study, we tested the effects of acetate on contractile function of isolated mouse ventricular myocytes. RESULTS: Acute exposure of myocytes to 10 mM sodium acetate caused a marked, but transient, decrease in systolic sarcomere shortening (1.49 ± 0.20% vs. 5.58 ± 0.49% in control), accompanied by a significant increase in diastolic sarcomere length (1.81 ± 0.01 µm vs. 1.77 ± 0.01 µm in control), with a near linear dose response in the 1-10 mM range. Unlike palmitate, acetate caused no change in action potential duration; however, acetate markedly increased mitochondrial Ca(2+) uptake. Moreover, pretreatment of cells with the mitochondrial Ca(2+) uptake blocker, Ru-360 (10 µM), markedly suppressed the effect of acetate on contraction. CONCLUSIONS: Lehninger and others have previously demonstrated that the anions of weak aliphatic acids such as acetate stimulate Ca(2+) uptake in isolated mitochondria. Here we show that this effect of acetate appears to extend to isolated cardiac myocytes where it transiently modulates cell contraction.

Promoter DNA methylation regulates murine SUR1 (Abcc8) and SUR2 (Abcc9) expression in HL-1 cardiomyocytes.

Two mammalian genes encode the SURx (SUR1, Abcc8 and SUR2, Abcc9) subunits that combine with Kir6.2 (Kcnj11) subunits to form the ATP-sensitive potassium (KATP) channel in cardiac myocytes. Different isoform combinations endow the channel with distinct physiological and pharmacological properties, and we have recently reported that the molecular composition of sarcolemmal KATP channels is chamber specific in the mouse heart. KATP channel composition is determined by what subunits are expressed in a cell or tissue. In the present study, we explore the role of CpG methylation in regulating SUR1 and SUR2 expression. In HL-1 cardiomyocytes, as in atrial myocytes, SUR1 expression is markedly greater than SUR2. Consistent with CpG methylation-dependent silencing of SUR2 expression, bisulfite sequencing of genomic DNA isolated from HL-1 cells demonstrates that 57.6% of the CpGs in the promoter region of the SUR2 gene are methylated, compared with 0.14% of the the CpG residues in the SUR1 sequence. Moreover, treatment with 10 µM 5-aza-2'-deoxycytidine (Aza-dC) significantly increased both the unmethylated fraction of the SUR2 CpG island and mRNA expression. However, we cannot rule out additional mechanisms of Aza-dC action, as Aza-dC also causes a decrease in SUR1 expression and lower doses of Aza-dC do not alter the unmethylated DNA fraction but do elicit a small increase in SUR2 expression. The conclusion that DNA methylation alone is not the only regulator of SUR subunit expression is also consistent with observations in native myocytes, where the CpG islands of both SUR genes are essentially unmethylated in both atrial and ventricular myocytes. Collectively, these data demonstrate the potential for CpG methylation to regulate SURx subunit expression and raises the possibility that regulated or aberrant CpG methylation might play a role in controlling channel structure and function under different physiological conditions or different species.

Increased dietary potassium and magnesium attenuate experimental volume dependent hypertension possibly through endogenous sodium-potassium pump inhibitor.

We and others have shown that inhibition of cardiovascular muscle (CVM) cell Na+,K-ATPase activity (NKPTA) due to increased level of endogenous sodium potassium pump inhibitor (SPI) is involved in the mechanism of volume expanded (VE) experimental and human essential hypertension (HT). Since diets fortified with very high potassium (K) or very high magnesium (Mg) decrease blood pressure (BP), we have examined the effect of a moderate increase in dietary K alone and a moderate increase in dietary K and Mg on plasma levels of SPI, CVM cell NKPTA, and BP in reduced renal mass (RRM)-salt HT rats, a classical model of VE HT. Seventy Percent-RRM rats were divided in four dietary groups, (1) Na free and normal K and Mg (0Na-K-Mg); (2) normal Na, K and Mg (Na-K-Mg); (3) normal Na and high K (2 x normal), and normal Mg (Na-2K-Mg); and (4) normal Na and high K (2 x normal), and high Mg (2 x normal) (Na-2K-2Mg). As expected, compared to control 0Na-K-Mg rats, Na-K-Mg rats developed HT. Blood pressure increased significantly less in Na-2K-Mg rats whereas, BP did not increase in Na-2K-2Mg rats. Hypertension in NA-K-Mg rats was associated with an increase in plasma SPI and digitalis like factor (DIF) and a decrease in renal and myocardial NKPTA. However, doubling the Mg along with K in the diet (Na-2K-2Mg) normalized SPI and DIF and increased myocardial and renal NKPTA, compared to control 0Na-K-Mg rats. Also, compared to 0Na-K-Mg rats, water consumption, urine excretion, urinary sodium excretion urinary potassium excretion (U(Na)V), and (U(K)V) increased in the other three groups, more so in Na-2K-2Mg rats. These data show that K and Mg have additive effects in preventing an increase in SPI, thus probably preventing the BP increase in RRM rats.