Search

Dilated cardiomyopathy (DCM) is clinically characterized by dilated ventricular cavities and reduced ejection fraction, leading to heart failure and increased thromboembolic risk. Mutations in thin-filament regulatory proteins can cause DCM and have been shown in vitro to reduce contractility and myofilament Ca2+-affinity. In this work we have studied the functional consequences of mutations in cardiac troponin T (R131W), cardiac troponin I (K36Q) and α-tropomyosin (E40K) using adenovirally transduced isolated guinea pig left ventricular cardiomyocytes. We find significantly reduced fractional shortening with reduced systolic Ca2+. Contraction and Ca2+ reuptake times were slowed, which contrast with some findings in murine models of myofilament Ca2+ desensitization. We also observe increased sarcoplasmic reticulum (SR) Ca2+ load and smaller fractional SR Ca2+ release. This corresponds to a reduction in SR Ca2+-ATPase activity and increase in sodium-calcium exchanger activity. We also observe dephosphorylation and nuclear translocation of the nuclear factor of activated T cells (NFAT), with concordant RAC-α-serine/threonine protein kinase (Akt) phosphorylation but no change to extracellular signal‐regulated kinase activation in chronically paced cardiomyocytes expressing DCM mutations. These changes in Ca2+ handling and signaling are common to all three mutations, indicating an analogous pathway of disease pathogenesis in thin-filament sarcomeric DCM. Previous work has shown that changes to myofilament Ca2+ sensitivity caused by DCM mutations are qualitatively opposite from hypertrophic cardiomyopathy (HCM) mutations in the same genes. However, we find several common pathways such as increased relaxation times and NFAT activation that are also hallmarks of HCM. This suggests more complex intracellular signaling underpinning DCM, driven by the primary mutation.NEW & NOTEWORTHY Dilated cardiomyopathy (DCM) is a frequently occurring cardiac disorder with a degree of genetic inheritance. We have found that DCM mutations in proteins that regulate the contractile machinery cause alterations to contraction, calcium-handling, and some new signaling pathways that provide stimuli for disease development. We have used guinea pig cells that recapitulate human calcium-handling and introduced the mutations using adenovirus gene transduction to look at the initial triggers of disease before remodeling.Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/dcm-mutations-alter-intracellular-ca2-and-signaling/. [ABSTRACT FROM AUTHOR]

Atrophic limbs exhibit decreased blood flow and histological changes in the arteries perfusing muscles. However, the effect of atrophy on vascular smooth muscle function is poorly understood. Here, we investigated the effect of unilateral sciatic denervation on the myogenic response (MR) and the ionic currents in deep femoral artery (DFA) smooth muscles from Sprague-Dawley rats. Because denervated rats were capable of treadmill exercise (20 m/min, 30 min, 3 times/wk), the impact of exercise training on these effects was also assessed. Skeletal arteries were harvested 3 or 5 wk after surgery. Then skeletal arteries or myocytes were subjected to video analysis of pressurized artery, myography, whole-cell patch clamp, and real-time quantitative PCR to determine the effect of hindlimb paralysis in the presence/absence of exercise training on MR, contractility, ionic currents, and channel transcription, respectively. In sedentary rats, atrophy was associated with loss of MR in the DFA at 5 wk. The contralateral DFA had a normal MR. At 5 wk after surgery, DFA myocytes from the atrophic limbs exhibited depressed L-type Ca2+ currents, GTP7S-induced transient receptor potential cation channel (TRPC)-like currents, 80 mM KCl- induced vasoconstriction, TRPC6 mRNA, and voltage-gated K+ and inwardly rectifying K+ currents. Exercise training abrogated the differences in all of these functions between atrophic side and contralateral side DFA myocytes. These results suggest that a probable increase in hemodynamic stimuli in skeletal artery smooth muscle plays an important role in maintaining MR and ionic currents in skeletal artery smooth muscle. This may also explain the observed benefits of exercise in patients with limb paralysis. NEW & NOTEWORTHY Myogenic responses (MRs) in rat skeletal arteries feeding the unilateral atrophic hindlimb were impaired. In addition, the L-type Ca2+ channel current, the TRPC6-like current, and TRPC6 mRNA levels in the corresponding myocytes decreased. Voltage-gated K+ channel currents and inwardly rectifying K+ channel currents were also attenuated in atrophic side myocytes. Exercise training effectively abrogated electrophysiological dysfunction of atrophic side myocytes and prevented loss of the MR. [ABSTRACT FROM AUTHOR]

TWIK-related two-pore domain K+ channels (TREKs) are activated by acidic intracellular pH (pHi), membrane stretch, temperature, and arachidonic acid (AA). Phosphatidylinositol 4,5-bisphosphate (PIP2) exerts concentration-dependent biphasic regulations, which have been observed: inhibition by high PIP2, activation by partial decrease of PIP2, and inhibition by depletion of PIP2. Consistently, the stimulation of voltage-sensitive PIP2 phosphatase (Dr-VSP) induces initial activation and subsequent inhibition of TREKs. Lys in the proximal C-terminus (pCt) is responsible for the inhibition by high PIP2, which is generated by phosphatidylinositol kinases with ATP; its neutralizing mutation [K330A of human TREK-2 (hTREK-2)] induces tonic high activity, irrespective of ATP. Here we focus on triple successive Arg in pCt (R3-pCt) as a candidate region for the stimulatory regulation by lower PIP2. Their neutralized mutant (R3A-pCt; RRR340-2A and RRR355-7A in hTREK-1 and -2, respectively) showed negligible basal current and was not affected by ATP removal or by Dr-VSP activation. Phosphatidic acid, a phospholipid agonist of TREKs, did not activate R3A-pCt. In contrast, acidic pHi, AA, and high temperature activated R3A-pCt normally, whereas activation by membrane stretch was attenuated. In hTREK-2, combined neutralizations of the inhibitory K330 and R3-pCt (K330A/RRR355-7A) did not recover the suppressed current. In contrast, combined neutralization of pHi-sensing Glu (E332A/R355-7A) induced tonic high current and no further activation by pHi. Interestingly, when the Gly between K330/E332 and R3-pCt was mutated (G334A), hTREK-2 was tonic activated with reversed responses to ATP and acidic pHi. Therefore, we propose that the PIP2-dependent converse regulation of TREKs by Lys and R3-pCt with Gly implies structural flexibility. [ABSTRACT FROM AUTHOR]

We previously reported that hypoxia augments α-adrenergic contraction (hypoxic vasoconstriction, HVC) of skeletal arteries in rats. The underlying mechanism may involve hypoxic inhibition of endothelial nitric oxide synthase (eNOS) expressed in skeletal arterial myocytes (16). To further explore the novel role of muscular eNOS in the skeletal artery, we compared HVC in femoral arteries (FAs) from eNOS knockout (KO) mice with that from wild-type (WT) and heterozygous (HZ) mice. Immunohistochemical assays revealed that, in addition to endothelia, eNOS is also expressed in the medial layer of FAs, albeit at a much lower level. However, the medial eNOS signal was not evident in HZ FAs, despite strong expression in the endothelium; similar observations were made in WT carotid arteries (CAs). The amplitude of contraction induced by 1 μM phenylephrine (PhE) was greater in HZ than in WT FAs. Hypoxia (3% PO2) significantly augmented PhE-induced contraction in WT FAs but not in HZ or KO FAs. No HVC was observed in PhE-pretreated WT CAs. The NOS inhibitor nitro-L-arginine methyl ester (0.1 mM) also augmented PhE contraction in endothelium-denuded WT FAs but not in WT CAs. Inhibitors specific to neuronal NOS and inducible NOS did not augment PhE-induced contraction of WT FAs. NADPH oxidase 4 (NOX4) inhibitor (GKT137831, 5 μM), but not NOX2 inhibitor (apocynin, 100 μM), suppressed HVC. Consistent with the role of reactive oxygen species (ROS), HVC was also inhibited by pretreatment with tiron or polyethylene glycol-catalase. Taken together, these data suggest that the eNOS expressed in smooth muscle cells in FAs attenuates α-adrenergic vasoconstriction; this suppression is alleviated under hypoxia, which potentiates vasoconstriction in a NOX4/ ROS-dependent mechanism. [ABSTRACT FROM AUTHOR]

In contrast to the conventional belief that systemic arteries dilate under hypoxia, we found that α-adrenergic contraction of rat deep femoral artery (DFA) is largely augmented by hypoxia (HVCDFA) while hypoxia (3% PO2) alone had no effect. HVCDFA was consistently observed in both endothelium-intact and -denuded vessels with partial pretone by phenylephrine (PhE) or by other conditions (e.g., K+ channel blocker). Patch-clamp study showed no change in the membrane conductance of DFA myocytes by hypoxia. The RhoA-kinase inhibitor Y27632 attenuated HVCDFA. The nitric oxide synthase inhibitor [nitro-L-arginine methyl ester (L-NAME)] and soluble guanylate cyclase inhibitor [oxadiazole quinoxalin (ODQ)] strongly augmented the PhE-pretone, while neither of the agents had effect without pretone. NADPH oxidase type 4 (NOX4) inhibitors (diphenylene iodonium and plumbagin) also potentiated PhE-pretone, which was reversed by NO donor. No additive HVCDFA was observed under the pretreatment with L-NAME, ODQ, or plumbagin. Western blot and immunohistochemistry analysis showed that both NOX4 and endothelial nitric oxide synthase (eNOS) are expressed in smooth muscle layer of DFA. Various mitochondria inhibitors (rotenone, myxothiazol, and cyanide) prevented HVCDFA. From the pharmacological data, as a mechanism for HVCDFA, we suggest hypoxic inhibition of eNOS in myocytes. The putative role of NOX4 and mitochondria requires further investigation. The HVCDFA may prevent imbalance between cardiac output and skeletal blood flow under emergent hypoxia combined with increased sympathetic tone. [ABSTRACT FROM AUTHOR]

Hypoxia-induced pulmonary vasoconstriction (HPV) is critical for matching of ventilation/perfusion in lungs. Although hypoxic inhibition of K+ channels has been a leading hypothesis for depolarization of pulmonary arterial smooth muscle cells (PASMCs) under hypoxia, pharmacological inhibition of K+ channels does not induce significant contraction in rat pulmonary arteries. Because a partial contraction by thromboxane A2 (TXA2) is required for induction of HPV, we hypothesize that TXA2 receptor (TP) stimulation might activate depolarizing nonselective cation channels (NSCs). Consistently, we found that 5-10 nM U46619, a stable agonist for TP, was indispensible for contraction of rat pulmonary arteries by 4-aminopyridine, a blocker of voltage-gated K+ channel (Kv). Whole cell voltage clamp with rat PASMC revealed that U46619 induced a NSC current (INSC,TXA2) with weakly outward rectifying current-voltage relation. INSC,TXA2 was blocked by ruthenium red (RR), an antagonist of the transient receptor potential vanilloid-related channel (TRPV) subfamily. 2-Aminoethoxydiphenyl borate, an agonist for TRPV1-3, consistently activated NSC channels in PASMCs. In contrast, agonists for TRPV1 (capsaicin), TRPV3 (camphor), or TRPV4 (α-PDD) rarely induced an increase in the membrane conductance of PASMCs. RT-PCR analysis showed the expression of transcripts for TRPV2 and -4 in rat PASMCs. Finally, it was confirmed that pretreatment with RR largely inhibited HPV in the presence of U46619. The pretreatment with agonists for TRPV1 (capsaicin) and TRPV4 (α-PDD) was ineffective as pretone agents for HPV. Taken together, it is suggested that the concerted effects of INSC,TXA2 activation and Kv inhibition under hypoxia induce membrane depolarization sufficient for HPV. TRPV2 is carefully suggested as the TXA2-activated NSC in rat PASMC. [ABSTRACT FROM AUTHOR]