Stimulation of ICa by basal PKA activity is facilitated by caveolin-3 in cardiac ventricular myocytes

Abstract

L-type Ca channels (LTCC), which play a key role in cardiac excitation–contraction coupling, are located predominantly at the transverse (t-) tubules in ventricular myocytes. Caveolae and the protein caveolin-3 (Cav-3) are also present at the t-tubules and have been implicated in localizing a number of signaling molecules, including protein kinase A (PKA) and β2-adrenoceptors. The present study investigated whether disruption of Cav-3 binding to its endogenous binding partners influenced LTCC activity. Ventricular myocytes were isolated from male Wistar rats and LTCC current (ICa) recorded using the whole-cell patch-clamp technique. Incubation of myocytes with a membrane-permeable peptide representing the scaffolding domain of Cav-3 (C3SD) reduced basal ICa amplitude in intact, but not detubulated, myocytes, and attenuated the stimulatory effects of the β2-adrenergic agonist zinterol on ICa. The PKA inhibitor H-89 also reduced basal ICa; however, the inhibitory effects of C3SD and H-89 on basal ICa amplitude were not summative. Under control conditions, myocytes stained with antibody against phosphorylated LTCC (pLTCC) displayed a striated pattern, presumably reflecting localization at the t-tubules. Both C3SD and H-89 reduced pLTCC staining at the z-lines but did not affect staining of total LTCC or Cav-3. These data are consistent with the idea that the effects of C3SD and H-89 share a common pathway, which involves PKA and is maximally inhibited by H-89, and suggest that Cav-3 plays an important role in mediating stimulation of ICa at the t-tubules via PKA-induced phosphorylation under basal conditions, and in response to β2-adrenoceptor stimulation.

Reduced density and altered regulation of rat atrial L-type Ca2+current in heart failure

“…Although both PKA and CaMKII have previously been shown to contribute to the basal regulation of ventricular I CaL , particularly at the t-tubule membrane (7)(8)(9)12), basal I CaL in atrial myocytes from either sham control or failing hearts in the present study was insensitive to inhibition of PKA or CaMKII. Notably, the absence of regulation of basal atrial I CaL by PKA in CAL hearts in the present study contrasts markedly with the increased PKA-dependent regulation of basal I CaL at the t-tubule of ventricular myocytes reported recently from the same hearts (9).…”

“…We have previously reported that left ventricular ejection fraction was reduced by 50% and left ventricular end-diastolic volume was increased by more than 100% in CAL compared with Sham for this group of animals (9). Atrial myocytes were isolated using our standard methods (6,8), following rapid excision of the heart under pentobarbital anesthesia. Isolated myocytes were stored for 2-10 h before use on the day of isolation.…”

“…The reduction in atrial I CaL in coronary artery ligation-induced heart failure has been suggested to be due to decreased cAMP-dependent basal regulation of L-type Ca 2ϩ channels (4, 19). However, although the cAMP-dependent protein kinase, PKA, has also been demonstrated to play an important role in the basal regulation of I CaL in ventricular myocytes (7,8), the constitutive regulation of ventricular I CaL by PKA appears to be increased in a coronary artery ligation model of heart failure (9). In addition, the reduction in atrial I CaL in patients with chronic AF has been associated with a reduction in calciumcalmodulin-dependent protein kinase II (CaMKII) activity and an increase in protein phosphatase activity in AF with…”

Targeting caveolin-3 for the treatment of diabetic cardiomyopathy

“…However, rather than a direct interaction between Cav3 and Ca v 1.2 (the ion channel subunit of LTCCs) it is thought that Cav3 serves to sequester PKA bringing it in close proximity to the ion channel and a number of proteins involved in the β 2 AR/cAMP pathway to form a macromolecular signalling complex. Building upon this study, Orchard and colleagues (Bryant, et al, 2014) have recently shown that disruption of Cav3 leads to a decrease in the amplitude of the calcium current in response to β 2 -adrenergic stimulation, linking Cav3 to PKA activity. However, the depression of I Ca,L was not consistent with perturbation of only a small sub-population of LTCCs, suggesting that this signalling pathway may have implications for LTCCs within the t-tubules and hence E-C coupling.…”

Calcineurin signaling in the heart: The importance of time and place

“…Furthermore, this increase is can be reversed by calcineurin inhibition but not PKA inhibition [167, 168]. The finding that PKA activation of I Ca,L is specific to T-tubules and is lost upon detubulation might explain this discrepancy [164, 169, 170]. One possibility is that modification of I Ca,L by calcineurin occurs throughout the cell but is masked by the opposing action of PKA in T-tubules.…”