Key points• In smooth muscles, the sensitivity of contraction to Ca 2+ can be increased by the phosphorylation of CPI-17 and MYPT1, resulting in the inhibition of myosin light chain phosphatase (MLCP).• Ca 2+ sensitization of smooth muscle contraction has typically been studied by immersing muscles in solutions containing contractile agonists.• However, stimulating muscles by bath-applied agonists may not be equivalent to neurotransmitter release because different post-junctional receptors may be activated in response to these different modes of stimulation.• In this study we found that a bath-applied cholinergic agonist activates Ca 2+ sensitization mechanisms in gastric fundus smooth muscles that are different than those of cholinergic neurotransmission. Electrical field stimulation (EFS) only increased CPI-17 phosphorylation, while bath-applied carbachol increased both CPI-17 and MYPT1 phosphorylation.• With the cholinesterase inhibitor neostigmine present, both CPI-17 and MYPT1 phosphorylation were increased by EFS.• In fundus muscles of W/W v mice which lack intramuscular interstitial cells of Cajal (ICC-IMs), EFS alone increased both CPI-17 and MYPT1 phosphorylation.• These findings indicate that ACh availability determines which Ca 2+ sensitization mechanisms are activated, and ICC-IMs regulate the access of ACh to smooth muscles.Abstract Ca 2+ sensitization of contraction has typically been investigated by bathing muscles in solutions containing agonists. However, it is unknown whether bath-applied agonists and enteric neurotransmission activate similar Ca 2+ sensitization mechanisms. We investigated protein kinase C (PKC)-potentiated phosphatase inhibitor protein of 17 kDa (CPI-17) and myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation in murine gastric fundus muscles stimulated by bath-applied carbachol (CCh) or cholinergic motor neurotransmission. CCh increased MYPT1 phosphorylation at Thr696 (pT696) and Thr853 (pT853), CPI-17 at Thr38 (pT38), and myosin light chain at Ser19 (pS19). Electrical field stimulation (EFS) only increased pT38. In the presence of neostigmine, EFS increased pT38, pT853 and pS19. In fundus muscles of W/W v mice, EFS alone increased pT38 and pT853. Atropine blocked all contractions and all increases in pT696, pT853, pT38 and pS19. The Rho kinase (ROCK) inhibitor SAR1x blocked increases in pT853 and pT696. The PKC inhibitors Go6976 and Gf109203x or nicardipine blocked increases in pT38 and pT696. These findings suggest that cholinergic motor neurotransmission activates PKC-dependent CPI-17 phosphorylation. Bath-applied CCh recruits additional ROCK-dependent MYPT1 phosphorylation due to exposure of the agonist to a wider population of muscarinic receptors. Intramuscular interstitial cells of Cajal (ICC-IMs) and cholinesterases restrict ACh accessibility to a select population of muscarinic receptors, possibly only those expressed by ICC-IMs. These results provide the first biochemical evidence for focalized (or synaptic-like) neurotransmission, rather than diffuse 'vo...

Rationale The cellular and molecular basis for post myocardial infarction (MI) structural and functional remodeling is not well understood. Objective To determine if Ca2+ influx through transient receptor potential (canonical) (TRPC) channels contributes to post-MI structural and functional remodeling. Methods and Results TRPC1/3/4/6 channel mRNA increased after MI in mice and was associated with TRPC-mediated Ca2+ entry. Cardiac myocyte specific expression of a dominant negative (dn: loss of function) TRPC4 channel increased basal myocyte contractility and reduced hypertrophy and cardiac structural and functional remodeling after MI while increasing survival. We used adenovirus-mediated expression of TRPC3/4/6 channels in cultured adult feline myocytes (AFMs) to define mechanistic aspects of these TRPC-related effects. TRPC3/4/6 over expression in AFMs induced calcineurin (Cn)-Nuclear Factor of Activated T cells (NFAT) mediated hypertrophic signaling, which was reliant on caveolae targeting of TRPCs. TRPC3/4/6 expression in AFMs increased rested state contractions and increased spontaneous sarcoplasmic reticulum (SR) Ca2+ sparks mediated by enhanced phosphorylation of the ryanodine receptor. TRPC3/4/6 expression was associated with reduced contractility and response to catecholamines during steady state pacing, likely due to enhanced SR Ca2+ leak. Conclusions Ca2+ influx through TRPC channels expressed after MI activates pathological cardiac hypertrophy and reduces contractility reserve. Blocking post-MI TRPC activity improved post-MI cardiac structure and function.