Contraction Rate Of Cardiomyocytes Research Articles

Cardiac striatin interacts with caveolin-3 and calmodulin in a calcium sensitive manner and regulates cardiomyocyte spontaneous contraction rate

Impaired cardiomyocyte contraction rate is detrimental to cardiac function and often lethal. Despite advancements in the field, there is a paucity of information regarding the coordination of molecules implicated in regulating the heart rate. Striatin (STRN) is a dynamic protein with binding domains to calmodulin (CaM) and caveolin (Cav), both of which are regulators of myocardial function. However, its role in cardiomyocyte contraction is not yet determined. Herein, we show that STRN is expressed in cardiomyocytes and is more abundant in atrial myocardium than in ventricles. Cardiac expression of STRN (protein and mRNA) was developmentally regulated with the highest expression being at neonatal stage (day one) and the lowest in adult rats (13 weeks). CaM pulldown assay indicated that the interaction of cardiac STRN with CaM and caveolin-3 (Cav-3) was calcium sensitive. Interestingly, the overexpression of STRN induced an increase (∼2-fold) in the rate of the spontaneous contraction of cultured cardiomyocytes, while the knockdown of STRN reduced their contraction rate (∼40%). The expression level of STRN was inversely proportional to the interaction of Cav-3 with the CaM/STRN complex. Collectively, our data delineate a novel role for STRN in regulating cardiomyocyte spontaneous contraction rate and the dynamics of the STRN/Cav-3/CaM complex.

Cardiac β2-Adrenergic Receptor Phosphorylation at Ser355/356 Regulates Receptor Internalization and Functional Resensitization.

Previous studies have demonstrated that β2-adrenergic receptors (β2ARs) can be phosphorylated by G protein-coupled receptor kinases (GRKs) and protein kinase A (PKA), affecting β2AR internalization and desensitization. However, the exact physiological function of β2ARs in cardiomyocytes is unknown. In this study, we showed that neonatal mouse cardiomyocytes had different contraction and internalization responses to sustained or repeated, transient agonist stimulation. Specifically, short-time stimulation (10 min) with epinephrine or norepinephrine increased the cardiomyocyte contraction rate, reaching a maximum at 5 min, followed by a slow decline. When the agonist was re-added after a 60-min wash-out period, the increase in the cardiomyocyte contraction rate was similar to the initial response. In contrast, when cardiomyocytes were exposed continuously to epinephrine or norepinephrine for 60 min, the second agonist stimulation did not increase the contraction response. These results indicated that continuous β2AR stimulation caused functional desensitization. Phosphorylation of β2ARs at serine (Ser)355/356 GRK phosphorylation sites, but not at Ser345/346 PKA phosphorylation sites increased with continuous epinephrine stimulation for 60 min. Accordingly, β2AR internalization increased. Interestingly, β2AR internalization was blocked by mutations at the GRK phosphorylation sites, but not by mutations at the PKA phosphorylation sites. Furthermore, inhibition of β2AR dephosphorylation by okadaic acid, a phosphatase 2A inhibitor, impaired the recovery of internalized β2ARs and reduced the cardiomyocyte contraction rate in response to epinephrine. Finally, epinephrine treatment induced the physical interaction of β-arrestin with internalized β2ARs in cardiomyocytes. Together, these data revealed the essential role of the Ser355/356 phosphorylation status of β2ARs in regulating receptor internalization and physiological resensitization in neonatal cardiomyocytes to contraction functions.

Ultrasound-induced modulation of cardiac rhythm in neonatal rat ventricular cardiomyocytes.

Isolated neonatal rat ventricular cardiomyocytes were used to study the influence of ultrasound on the chronotropic response in a tissue culture model. The beat frequency of the cells, varying from 40 to 90 beats/min, was measured based upon the translocation of the nuclear membrane captured by a high-speed camera. Ultrasound pulses (frequency = 2.5 MHz) were delivered at 300-ms intervals [3.33 Hz pulse repetition frequency (PRF)], in turn corresponding to 200 pulses/min. The intensity of acoustic energy and pulse duration were made variable, 0.02-0.87 W/cm(2) and 1-5 ms, respectively. In 57 of 99 trials, there was a noted average increase in beat frequency of 25% with 8-s exposures to ultrasonic pulses. Applied ultrasound energy with a spatial peak time average acoustic intensity (Ispta) of 0.02 W/cm(2) and pulse duration of 1 ms effectively increased the contraction rate of cardiomyocytes (P < 0.05). Of the acoustic power tested, the lowest level of acoustic intensity and shortest pulse duration proved most effective at increasing the electrophysiological responsiveness and beat frequency of cardiomyocytes. Determining the optimal conditions for delivery of ultrasound will be essential to developing new models for understanding mechanoelectrical coupling (MEC) and understanding novel nonelectrical pacing modalities for clinical applications.

Striatin: a novel regulator of cardiomyocyte calcium homeostasis and contraction (864.3)

Although cardiac arrhythmias are still a major health threat for human, there is paucity of information on the molecular mechanisms involved in this pathology. Striatin (STRN) is a multivalent protein with dynamic domains including caveolin (Cav), Calmodulin (CaM), and Protein Phosphatase 2A (PP2A) binding sites, all of which are important regulatory units of the L‐type Calcium Channels (LCC) in cardiomyocytes. Previous studies indicate that mutations/deletions of the striatin gene correlate with the development of cardiac arrhythmias in animal models. We aimed to investigate whether STRN regulates calcium homeostasis and cardiomyocyte contraction rate. Data indicate that STRN is highly expressed in atrial myocardium when compared to the ventricles. Overexpression of STRN elevated the contraction rate (~ 3 fold) and resulted in sustained levels of KCl‐induced increases in intracellular calcium in neonatal rat cardiomyocytes. Conversely, silencing (&gt;80%) the STRN gene using shRNA was associated with decreased contraction rate (~50%) of cardiomyocytes. Interestingly, pharmacological challenge with isoproterenol (β‐adrenergic agonist) failed to improve the contraction rate of cardiomyocytes with low levels of striatin (shRNA‐STRN). Biochemical analysis (CaM pull down) revealed that the interaction between CaM/PP2A/Cav‐3/LCC was increased in shRNA‐STRN cardiomyocytes and reduced when overexpressing STRN. Collectively our data indicate that striatin is a novel regulator of cardiomyocyte calcium homeostasis and contraction rate by modulating the CaM/PP2A/Cav‐3/LCC complex and influencing the activity of the LCC in the myocardium.Grant Funding Source: King Faisal Specialist Hospital and Research Center

MRP4 and CFTR in the regulation of cAMP and β-adrenergic contraction in cardiac myocytes

Spatiotemporal regulation of cAMP in cardiac myocytes is integral to regulating the diverse functions downstream of β-adrenergic stimulation. The activities of cAMP phosphodiesterases modulate critical and well-studied cellular processes. Recently, in epithelial and smooth muscle cells, it was found that the multi-drug resistant protein 4 (MRP4) acts as a cAMP efflux pump to regulate intracellular cAMP levels and alter effector function, including activation of the cAMP-stimulated Cl− channel, CFTR (cystic fibrosis transmembrane conductance regulator). In the current study we investigated the potential role of MRP4 in regulating intracellular cAMP and β-adrenergic stimulated contraction rate in cardiac myocytes. Cultured neonatal ventricular myocytes were used for all experiments. In addition to wildtype mice, β1-, β2-, and β1/β2-adrenoceptor, and CFTR knockout mice were used. MRP4 expression was probed via Western blot, intracellular cAMP was measured by fluorescence resonance energy transfer, while the functional role of MRP4 was assayed via monitoring of isoproterenol-stimulated contraction rate. We found that MRP4 is expressed in mouse neonatal ventricular myocytes. A pharmacological inhibitor of MRP4, MK571, potentiated submaximal isoproterenol-stimulated cAMP accumulation and cardiomyocyte contraction rate via β1-adrenoceptors. CFTR expression was critical for submaximal isoproterenol-stimulated contraction rate. Interestingly, MRP4-dependent changes in contraction rate were CFTR-dependent, however, PDE4-dependent potentiation of contraction rate was CFTR-independent. We have shown, for the first time, a role for MRP4 in the regulation of cAMP in cardiac myocytes and involvement of CFTR in β-adrenergic stimulated contraction. Together with phosphodiesterases, MRP4 must be considered when examining cAMP regulation in cardiac myocytes.

Role of neuronal potassium M-channels in sympathetic regulation of cardiac function

Role of neuronal potassium M-channels in sympathetic regulation of cardiac function

Functional role of M-type (KCNQ) K+channels in adrenergic control of cardiomyocyte contraction rate by sympathetic neurons

M-type (KCNQ) K⁺ channels are known to regulate excitability and firing properties of sympathetic neurons (SNs), but their role in regulating neurotransmitter release is unclear, requiring further study. We sought to use a physiological preparation in which SNs innervate primary cardiomyocytes to evaluate the direct role of M-channels in the release of noradrenaline (NA) from SNs. Co-cultures of rat SNs and mouse cardiomyocytes were prepared, and the contraction rate (CR) of the cardiomyocyte syncytium monitored by video microscopy. We excited the SNs with nicotine, acting on nicotinic acetylcholine receptors, and monitored the increase in CR in the presence or absence of the specific M-channel opener retigabine, or agonists of bradykinin B2 or purinergic P2Y receptors on the SNs. The maximal adrenergic effect on the CR was determined by application of isoproterenol (isoprenaline). To isolate the actions of B2 or P2Y receptor stimulation to the neurons, we prepared cardiomyocytes from B2 receptor or P2Y2 receptor knock-out mice, respectively. We found that co-application of retigabine strongly decreased the nicotine-induced increase in CR. Conversely, co-application of bradykinin or the P2Y-receptor agonist UTP augmented the nicotine-induced increase in CR to about half of the level produced by isoproterenol. All effects on the CR were wholly blocked by propranolol. Our data support the role of M-type K⁺ channels in the control of NA release by SNs at functional adrenergic synapses on cardiomyocytes.We conclude that physiological receptor agonists control the heart rate via the regulation of M-current in SNs.

Berberine possesses muscarinic agonist-like properties in cultured rodent cardiomyocytes

Berberine, a natural product alkaloid, has been shown to display a wide array of pharmacological effects. Generally, the mechanism of action of each of these effects has not been well described. The aim of the present study is to test the hypothesis that some of berberine's cardiovascular effects are mediated through activation of cardiac M2 muscarinic cholinergic receptors. In our studies, we tested the ability of berberine to alter the contraction rate of cultured neonatal rodent cardiomyocytes. In these spontaneously contracting primary cultured cells, berberine reduced the contraction rate in a manner independent of β-adrenergic receptor blockade but sensitive to pertussis toxin, a Gi/o G protein inhibitor. Muscarinic antagonists completely blocked the effect of berberine on contraction rate of cardiomyocytes, whereas the effect of berberine was not opposed by antagonists to opioid, adenosine or α-adrenergic receptors. Further, berberine bound to muscarinic receptors of adult mouse heart membranes with relatively high affinity ( K i = 5.4 × 10 −6 M) comparable to that of the classic muscarinic agonist, carbachol, and to muscarinic M2 receptors exogenously expressed in HEK 293 cells ( K i = 4.9 × 10 −6 M). Therefore, the findings of the present study suggest that berberine is a muscarinic agonist at M2 receptors, potentially explaining some of its reported cardiovascular effects.

Cardiomyocytes with disrupted CFTR function require CaMKII and Ca2+‐activated Cl− channel activity to maintain contraction rate

The physiological role of the cystic fibrosis transmembrane conductance regulator (CFTR) in cardiomyocytes remains unclear. Using spontaneously beating neonatal ventricular cardiomyocytes from wild-type (WT) or CFTR knockout (KO) mice, we examined the role of CFTR in the modulation of cardiomyocyte contraction rate. Contraction rates of spontaneously beating myocytes were captured by video imaging. Real-time changes in intracellular ([Ca(2+)](i)) and protein kinase A (PKA) activity were measured by fura-2 and fluorescence resonance energy transfer, respectively. Acute inhibition of CFTR in WT cardiomyocytes using the CFTR inhibitor CFTR(inh)-172 transiently inhibited the contraction rate. By contrast, cardiomyocytes from CFTR KO mice displayed normal contraction rates. Further investigation revealed that acute inhibition of CFTR activity in WT cardiomyocytes activated L-type Ca(2+) channels, leading to a transient increase of [Ca(2+)](i) and inhibition of PKA activity. Additionally, we found that contraction rate normalization following acute CFTR inhibition in WT cardiomyocytes or chronic deletion in cardiomyocytes from CFTR KO mice requires the activation of Ca(2+)/calmodulin-dependent kinase II (CaMKII) and Ca(2+)-activated Cl(-) channels (CaCC) because simultaneous addition of myristoylated-autocamtide-2-related inhibitory peptide or niflumic acid and CFTR(inh)-172 to WT cardiomyocytes or treatment of cardiomyoctes from CFTR KO mice with these agents caused sustained attenuation of contraction rates. Our results demonstrate that regulation of cardiomyocyte contraction involves CFTR. They also reveal that activation of CaMKII and CaCC compensates for loss of CFTR function. Increased dependence on CaMKII upon loss of CFTR function might leave cystic fibrosis patients at increased risk of heart dysfunction and disease.

Modulation of CFTR activity alters submaximal β‐adrenergic receptor stimulated cardiomyocyte contraction rate

Previously we found that CFTR regulates the contraction rate of spontaneously beating neonatal ventricular cardiomyocytes. In this study we examined if CFTR is involved in β‐adrenergic receptor (β‐AR) stimulated increases in contraction rate. Spontaneous beating cardiomyocytes were generated by isolation and culture of mouse neonatal ventricular myocytes. Isoproterenol dose‐dependently increased contraction rates with an EC50 of 16.6 nM. Inhibition or knockout of CFTR led to a loss of submaximal β‐AR stimulation, resulting in a right‐shift of the dose response curve with an EC50 of 66.7 nM and 31.2 nM, respectively. This targeted response to submaximal β‐AR stimulated contraction rates was specific to CFTR as inhibition of volume‐ or Ca2+‐activated Cl− channels affected both submaximal and maximal concentrations. Furthermore, pre‐activation of CFTR with genistein or PG‐01 potentiated submaximal isoproterenol stimulated contraction rates, resulting in a left‐shift of the dose response curve with an EC50 of 1.1 nM and 0.6 nM, respectively. Our results indicate that CFTR plays a distinct role in regulating contraction rate responses to submaximal, but not maximal, β‐AR stimulation. Future work will be necessary to determine if individuals with alterations in CFTR activity (i.e. cystic fibrosis patients) may be at increased risk for cardiac abnormalities. Funded by the AHA, NIH, University of Illinois.

Regulation of Basal and Beta‐Adrenergic‐Stimulated Ventricular Cardiomyocyte Contraction Rate by Distinct Populations of Chloride Channels

A variety of Cl− channels have been identified in ventricular cardiomyocytes and are presumed to be important in modulation of cardiac action potential duration. In the current study we examined if modulation of Cl− channel activity affects ventricular cardiomyocyte contraction rate. Ventricular myocytes were isolated from neonatal mice and cultured until they formed a functional syncytium. Contraction rates were captured by video imaging and analyzed in the presence and absence of beta‐adrenergic receptor stimulation by isoproteronol (ISO). Cl− channel activity was inhibited by various Cl− channel inhibitors. Glibenclamide, DPC, and CFTRinh‐172 significantly, but transiently, inhibited baseline contraction rate. All three drugs significantly inhibited prolonged increases in ISO‐stimulated cardiomyocyte contraction rate. 9‐AC elicited a prolonged inhibitory effect on baseline contraction rate, but failed to significantly alter ISO‐stimulated contraction rate. Tamoxifen transiently increased baseline contraction rate, but significantly inhibited ISO‐stimulated increases in contraction rate. Inhibitors of CFTR, calcium‐activated, and swelling‐activated Cl− channels modulate ventricular cardiomyocyte contraction rate, but do so in distinct ways. Our results indicate an important role for CFTR in the regulation of cardiomyocyte contraction rate. Supported by a GIA from the AHA.

Norepinephrine- and Epinephrine-induced Distinct β2-Adrenoceptor Signaling Is Dictated by GRK2 Phosphorylation in Cardiomyocytes

Agonist-dependent activation of G protein-coupled receptors induces diversified receptor cellular and signaling properties. Norepinephrine (NE) and epinephrine (Epi) are two endogenous ligands that activate adrenoceptor (AR) signals in a variety of physiological stress responses in animals. Here we use cardiomyocyte contraction rate response to analyze the endogenous beta(2)AR signaling induced by Epi or NE in cardiac tissue. The Epi-activated beta(2)AR induced a rapid contraction rate increase that peaked at 4 min after stimulation. In contrast, the NE-activated beta(2)AR induced a much slower contraction rate increase that peaked at 10 min after stimulation. Whereas both drugs activated beta(2)AR coupling to G(s) proteins, only Epi-activated receptors were capable of coupling to G(i) proteins. Subsequent studies showed that the Epi-activated beta(2)AR underwent a rapid phosphorylation by G protein-coupled receptor kinase 2 (GRK2) and subsequent dephosphorylation on serine residues 355 and 356, which was critical for sufficient receptor recycling and G(i) coupling. In contrast, the NE-activated beta(2)ARs underwent slow GRK2 phosphorylation, receptor internalization and recycling, and failed to couple to G(i). Moreover, inhibiting beta(2)AR phosphorylation by betaARK C terminus or dephosphorylation by okadaic acid prevented sufficient recycling and G(i) coupling. Together, our data revealed that distinct temporal phosphorylation of beta(2)AR on serine 355 and 356 by GRK2 plays a critical role for dictating receptor cellular events and signaling properties induced by Epi or NE in cardiomyocytes. This study not only helps us understand the endogenous agonist-dependent beta(2)AR signaling in animal heart but also offers an example of how G protein-coupled receptor signaling may be finely regulated by GRK in physiological settings.