Molecular and Functional Identification of m ₁ Muscarinic Acetylcholine Receptors in Rat Ventricular Myocytes

Abstract

The expression of muscarinic acetylcholine receptor (mAChR) subtypes in freshly isolated adult rat ventricular myocytes was investigated by reverse transcription of cellular mRNA followed by amplification of cDNA using the polymerase chain reaction (PCR). After reverse-transcriptase PCR, bands were obtained corresponding to the expected sizes for the m1 and m2 but not for the m3 to m5 mAChRs. The identity of the m1 and m2 bands was confirmed by single-cell PCR, restriction digest mapping, and Southern blot analysis. The presence of m1 and m2, but not m3, mAChR protein in these cells was shown by indirect immunofluorescence studies using subtype-specific antibodies. It was further investigated whether the identified m1 mAChR was responsible for the stimulatory effects on Ca2+ transients by high concentrations of carbachol ( > 10 mumol/L) known to occur in these cells. In pertussis toxin-treated ventricular myocytes electrically stimulated at 1 Hz, carbachol (300 mumol/L) increased the basal Ca2+ level from 96 +/- 7 to 118 +/- 8 nmol/L and the peak Ca2+ transient level from 519 +/- 32 to 640 +/- 36 nmol/L (mean +/- SEM P < .05 for both, n = 8). These effects of carbachol on Ca2+ transients were antagonized by 10 nmol/L pirenzepine, an m1 mAChR-selective antagonist. In contrast, the m2 mAChR-selective antagonist methoctramine (up to 100 nmol/L) did not inhibit the response. These results are the first to use single-cell PCR to probe cardiomyocyte-specific gene expression and indicate that m1 mAChRs are expressed on adult rat ventricular myocytes in addition to m2 mAChRs. The results further suggest that m1 mAChRs mediate the stimulatory responses on Ca2+ transients to high concentrations of cholinergic agonists seen in these cells.

ASK1 Associates with Troponin T and Induces Troponin T Phosphorylation and Contractile Dysfunction in Cardiomyocytes

“…To determine effect of ASK1 expression on cardiac contractility, ASK1-expressing adult cardiac myocytes were field-stimulated at 1 Hz and Ca 2ϩ transients were measured as previously described. 34 Results show that ASK1 expression significantly inhibited both the rate and amplitude of Ca 2ϩ transients ( Figure 6E). These data strongly suggest that ASK1 activation induces cardiac contractile dysfunction.…”

“…34 Briefly, cultured rat adult cardiomyocytes were rinsed in PBS and fixed for 15 minutes in 3% formaldehyde. The cells were then permeabilized for 15 minutes with 0.2% Triton X-100 followed by drying on a glass slide.…”

“…34 Briefly, isolated rat adult myocytes were loaded for 15 minutes at room temperature with 1 mol/L of the fluorescent Ca 2ϩ indicator fura 2-AM (Molecular Probes) in HEPES-buffered solution containing (mmol/L) KCl 5, NaCl 140, CaCl 2 2, MgCl 2 1, glucose 10, and HEPES 10 (pH 7.4). The coverslip with dyeloaded cells was mounted in a tissue chamber (Bellco) on the stage of a Nikon Diaphot inverted microscope equipped for epifluorescence (Deltascan 1, Photon Technology International).…”

GIT1 Mediates Src-dependent Activation of Phospholipase Cγ by Angiotensin II and Epidermal Growth Factor

“…The samples were then measured according to the manufacturer's protocol (Amersham Biosciences). Intracellular Ca 2ϩ was measured as previously described (39). In brief, cells were grown on glass coverslips.…”

Dynamic Targeting of the Agonist-stimulated m2 Muscarinic Acetylcholine Receptor to Caveolae in Cardiac Myocytes

“…Recent studies have shown that, of the five mAchR subtypes identified to date, only the m1 and m2 subtypes are expressed in adult mammalian cardiac tissues (3,4). According to these reports, the m2 mAchR, which is expressed at a much higher level than the m1 mAchR, triggers the inhibitory response while m1 receptor activation elicits, when stimulated by higher concentrations of agonist, a compensatory excitatory effect on heart function.…”