Alexander Katchman

Protein kinase C isoforms differentially phosphorylate Ca(v)1.2 alpha(1c).

The regulation of Ca(2+) influx through the phosphorylation of the L-type Ca(2+) channel, Ca(v)1.2, is important for the modulation of excitation-contraction (E-C) coupling in the heart. Ca(v)1.2 is thought to be the target of multiple kinases that mediate the signals of both the renin-angiotensin and sympathetic nervous systems. Detailed biochemical information regarding the protein phosphorylation reactions involved in the regulation of Ca(v)1.2 is limited. The protein kinase C (PKC) family of kinases can modulate cardiac contractility in a complex manner, such that contractility is either enhanced or depressed and relaxation is either accelerated or slowed. We have previously reported that Ser(1928) in the C-terminus of alpha(1c) was a target for PKCalpha, -zeta, and -epsilon phosphorylation. Here, we report the identification of seven PKC phosphorylation sites within the alpha(1c) subunit. Using phospho-epitope specific antibodies to Ser(1674) and Ser(1928), we demonstrate that both sites within the C-terminus are phosphorylated in HEK cells in response to PMA. Phosphorylation was inhibited with a PKC inhibitor, bisindolylmaleimide. In Langendorff-perfused rat hearts, both Ser(1674) and Ser(1928) were phosphorylated in response to PMA. Phosphorylation of Ser(1674), but not Ser(1928), is PKC isoform specific, as only PKCalpha, -betaI, -betaII, -gamma, -delta, and -theta, but not PKCepsilon, -zeta, and -eta, were able to phosphorylate this site. Our results identify a molecular mechanism by which PKC isoforms can have different effects on channel activity by phosphorylating different residues.

Cardiac L-type calcium channel (Cav1.2) associates with γ subunits

The cardiac voltage‐gated Ca2+ channel, Cav1.2, mediates excitation‐contraction coupling in the heart. The molecular composition of the channel includes the pore‐forming α1 subunit and auxiliary α2/δ‐1 and β subunits. Ca2+ channel γ subunits, of which there are 8 isoforms, consist of 4 transmembrane domains with intracellular N‐ and C‐terminal ends. The γ1 subunit was initially detected in the skeletal muscle Cav1.1 channel complex, modulating current amplitude and activation and inactivation properties. The γ1 subunit also shifts the steady‐state inactivation to more negative membrane potentials, accelerates current inactivation, and increases peak currents, when coexpressed with the cardiac α1c subunit in Xenopus oocytes and human embryonic kidney (HEK) 293 cells. The γ1 subunit is not expressed, however, in cardiac muscle. We sought to determine whether γ subunits that are expressed in cardiac tissue physically associate with and modulate Cav1.2 function. We now demonstrate that γ4, γ6, γ7, and γ8 subunits physically interact with the Cav1.2 complex. The γ subunits differentially modulate Ca2+ channel function when coexpressed with the β1b and α2/δ‐1 subunits in HEK cells, altering both activation and inactivation properties. The effects of γ on Cav1.2 function are dependent on the subtype of β subunit. Our results identify new members of the cardiac Cav1.2 macro‐molecular complex and identify a mechanism by which to increase the functional diversity of Cav1.2 channels.—Yang, L., Katchman, A., Morrow, J. P., Doshi, D., Marx, S. A. Cardiac L‐type calcium channel (CaV1.2) associates with γ subunits. FASEB J. 25, 928–936 (2011). www.fasebj.org

β-Adrenergic Regulation of the L-type Ca2+ Channel does not Require Phosphorylation of α1C Ser1700

Rationale: Sympathetic nervous system triggered activation of protein kinase A, which phosphorylates several targets within cardiomyocytes, augments inotropy, chronotropy, and lusitropy. An important target of &bgr;-adrenergic stimulation is the sarcolemmal L-type Ca2+ channel, CaV1.2, which plays a key role in cardiac excitation–contraction coupling. The molecular mechanisms of &bgr;-adrenergic regulation of CaV1.2 in cardiomyocytes, however, are incompletely known. Recently, it has been postulated that proteolytic cleavage at Ala1800 and protein kinase A phosphorylation of Ser1700 are required for &bgr;-adrenergic modulation of CaV1.2. Objective: To assess the role of Ala1800 in the cleavage of &agr;1C and the role of Ser1700 and Thr1704 in mediating the adrenergic regulation of CaV1.2 in the heart. Methods and Results: Using a transgenic approach that enables selective and inducible expression in mice of FLAG-epitope–tagged, dihydropyridine-resistant CaV1.2 channels harboring mutations at key regulatory sites, we show that adrenergic regulation of CaV1.2 current and fractional shortening of cardiomyocytes do not require phosphorylation of either Ser1700 or Thr1704 of the &agr;1C subunit. The presence of Ala1800 and the 1798NNAN1801 motif in &agr;1C is not required for proteolytic cleavage of the &agr;1C C-terminus, and deletion of these residues did not perturb adrenergic modulation of CaV1.2 current. Conclusions: These results show that protein kinase A phosphorylation of &agr;1C Ser1700 does not have a major role in the sympathetic stimulation of Ca2+ current and contraction in the adult murine heart. Moreover, this new transgenic approach enables functional and reproducible screening of &agr;1C mutants in freshly isolated adult cardiomyocytes in a reliable, timely, cost-effective manner.

Mice With Cardiac Overexpression of Peroxisome Proliferator–Activated Receptor γ Have Impaired Repolarization and Spontaneous Fatal Ventricular Arrhythmias

Background— Diabetes mellitus and obesity, which confer an increased risk of sudden cardiac death, are associated with cardiomyocyte lipid accumulation and altered cardiac electric properties, manifested by prolongation of the QRS duration and QT interval. It is difficult to distinguish the contribution of cardiomyocyte lipid accumulation from the contribution of global metabolic defects to the increased incidence of sudden death and electric abnormalities. Methods and Results— In order to study the effects of metabolic abnormalities on arrhythmias without the complex systemic effects of diabetes mellitus and obesity, we studied transgenic mice with cardiac-specific overexpression of peroxisome proliferator–activated receptor &ggr; 1 (PPAR&ggr;1) via the cardiac &agr;-myosin heavy-chain promoter. The PPAR&ggr; transgenic mice develop abnormal accumulation of intracellular lipids and die as young adults before any significant reduction in systolic function. Using implantable ECG telemeters, we found that these mice have prolongation of the QRS and QT intervals and spontaneous ventricular arrhythmias, including polymorphic ventricular tachycardia and ventricular fibrillation. Isolated cardiomyocytes demonstrated prolonged action potential duration caused by reduced expression and function of the potassium channels responsible for repolarization. Short-term exposure to pioglitazone, a PPAR&ggr; agonist, had no effect on mortality or rhythm in WT mice but further exacerbated the arrhythmic phenotype and increased the mortality in the PPAR&ggr; transgenic mice. Conclusions— Our findings support an important link between PPAR&ggr; activation, cardiomyocyte lipid accumulation, ion channel remodeling, and increased cardiac mortality.Background Diabetes and obesity, which confer an increased risk of sudden cardiac death, are associated with cardiomyocyte lipid accumulation and altered cardiac electrical properties, manifested by prolongation of the QRS duration and QT interval. It is difficult to distinguish the contribution of cardiomyocyte lipid accumulation versus the contribution of global metabolic defects to the increased incidence of sudden death and electrical abnormalities.

Aberrant sodium influx causes cardiomyopathy and atrial fibrillation in mice

Increased sodium influx via incomplete inactivation of the major cardiac sodium channel Na(V)1.5 is correlated with an increased incidence of atrial fibrillation (AF) in humans. Here, we sought to determine whether increased sodium entry is sufficient to cause the structural and electrophysiological perturbations that are required to initiate and sustain AF. We used mice expressing a human Na(V)1.5 variant with a mutation in the anesthetic-binding site (F1759A-Na(V)1.5) and demonstrated that incomplete Na+ channel inactivation is sufficient to drive structural alterations, including atrial and ventricular enlargement, myofibril disarray, fibrosis and mitochondrial injury, and electrophysiological dysfunctions that together lead to spontaneous and prolonged episodes of AF in these mice. Using this model, we determined that the increase in a persistent sodium current causes heterogeneously prolonged action potential duration and rotors, as well as wave and wavelets in the atria, and thereby mimics mechanistic theories that have been proposed for AF in humans. Acute inhibition of the sodium-calcium exchanger, which targets the downstream effects of enhanced sodium entry, markedly reduced the burden of AF and ventricular arrhythmias in this model, suggesting a potential therapeutic approach for AF. Together, our results indicate that these mice will be important for assessing the cellular mechanisms and potential effectiveness of antiarrhythmic therapies.

The PDZ Motif of the α1C Subunit Is Not Required for Surface Trafficking and Adrenergic Modulation of CaV1.2 Channel in the Heart

Background: The mechanisms responsible for CaV1.2 regulation by the α1C C terminus are unknown. Results: Trafficking, basal function, and adrenergic modulation of CaV1.2 were not altered in cardiomyocytes of transgenic mice expressing PDZ-deleted α1C. Conclusion: PDZ-mediated interactions are not required for CaV1.2 trafficking and function in the heart. Significance: The regulation of CaV1.2 by auxiliary proteins does not depend on the PDZ ligand motif in the heart. Voltage-gated Ca2+ channels play a key role in initiating muscle excitation-contraction coupling, neurotransmitter release, gene expression, and hormone secretion. The association of CaV1.2 with a supramolecular complex impacts trafficking, localization, turnover, and, most importantly, multifaceted regulation of its function in the heart. Several studies hint at an important role for the C terminus of the α1C subunit as a hub for multidimensional regulation of CaV1.2 channel trafficking and function. Recent studies have demonstrated an important role for the four-residue PDZ binding motif at the C terminus of α1C in interacting with scaffold proteins containing PDZ domains, in the subcellular localization of CaV1.2 in neurons, and in the efficient signaling to cAMP-response element-binding protein in neurons. However, the role of the α1C PDZ ligand domain in the heart is not known. To determine whether the α1C PDZ motif is critical for CaV1.2 trafficking and function in cardiomyocytes, we generated transgenic mice with inducible expression of an N-terminal FLAG epitope-tagged dihydropyridine-resistant α1C with the PDZ motif deleted (ΔPDZ). These mice were crossed with α-myosin heavy chain reverse transcriptional transactivator transgenic mice, and the double-transgenic mice were fed doxycycline. The ΔPDZ channels expressed, trafficked to the membrane, and supported robust excitation-contraction coupling in the presence of nisoldipine, a dihydropyridine Ca2+ channel blocker, providing functional evidence that they appropriately target to dyads. The ΔPDZ Ca2+ channels were appropriately regulated by isoproterenol and forskolin. These data indicate that the α1C PDZ motif is not required for surface trafficking, localization to the dyad, or adrenergic stimulation of CaV1.2 in adult cardiomyocytes.

Proteolytic cleavage and PKA phosphorylation of α1C subunit are not required for adrenergic regulation of CaV1.2 in the heart

Significance Calcium influx through the cardiac voltage-dependent L-type calcium channel (CaV1.2) increases during “fight or flight” through activation of the β-adrenergic and protein kinase A (PKA) signaling pathway. None of the previously identified sites in the α1C subunit, each painstakingly and singly investigated, were shown to be required for adrenergic modulation of CaV1.2. Our approach allowed an unprecedented and massive increase in throughput, because we mutated many potential PKA phosphorylation sites throughout α1C. By creating transgenic mice expressing either α1C with alanines substituted for all conserved consensus PKA phosphorylation sites or, separately, α1C without C-terminal proteolytic cleavage, our paradigm-shifting results show that acute β-adrenergic regulation of CaV1.2 does not require phosphorylation of any conserved Ser/Thr of α1C or the proteolytic cleavage of the C terminus of α1C. Calcium influx through the voltage-dependent L-type calcium channel (CaV1.2) rapidly increases in the heart during “fight or flight” through activation of the β-adrenergic and protein kinase A (PKA) signaling pathway. The precise molecular mechanisms of β-adrenergic activation of cardiac CaV1.2, however, are incompletely known, but are presumed to require phosphorylation of residues in α1C and C-terminal proteolytic cleavage of the α1C subunit. We generated transgenic mice expressing an α1C with alanine substitutions of all conserved serine or threonine, which is predicted to be a potential PKA phosphorylation site by at least one prediction tool, while sparing the residues previously shown to be phosphorylated but shown individually not to be required for β-adrenergic regulation of CaV1.2 current (17-mutant). A second line included these 17 putative sites plus the five previously identified phosphoregulatory sites (22-mutant), thus allowing us to query whether regulation requires their contribution in combination. We determined that acute β-adrenergic regulation does not require any combination of potential PKA phosphorylation sites conserved in human, guinea pig, rabbit, rat, and mouse α1C subunits. We separately generated transgenic mice with inducible expression of proteolytic-resistant α1C. Prevention of C-terminal cleavage did not alter β-adrenergic stimulation of CaV1.2 in the heart. These studies definitively rule out a role for all conserved consensus PKA phosphorylation sites in α1C in β-adrenergic stimulation of CaV1.2, and show that phosphoregulatory sites on α1C are not redundant and do not each fractionally contribute to the net stimulatory effect of β-adrenergic stimulation. Further, proteolytic cleavage of α1C is not required for β-adrenergic stimulation of CaV1.2.

Mice with cardiac overexpression of PPARγ have impaired repolarization and spontaneous fatal ventricular arrhythmias (Morrow, PPARγ overexpression induces fatal arrhythmias)

Background— Diabetes mellitus and obesity, which confer an increased risk of sudden cardiac death, are associated with cardiomyocyte lipid accumulation and altered cardiac electric properties, manifested by prolongation of the QRS duration and QT interval. It is difficult to distinguish the contribution of cardiomyocyte lipid accumulation from the contribution of global metabolic defects to the increased incidence of sudden death and electric abnormalities. Methods and Results— In order to study the effects of metabolic abnormalities on arrhythmias without the complex systemic effects of diabetes mellitus and obesity, we studied transgenic mice with cardiac-specific overexpression of peroxisome proliferator–activated receptor &ggr; 1 (PPAR&ggr;1) via the cardiac &agr;-myosin heavy-chain promoter. The PPAR&ggr; transgenic mice develop abnormal accumulation of intracellular lipids and die as young adults before any significant reduction in systolic function. Using implantable ECG telemeters, we found that these mice have prolongation of the QRS and QT intervals and spontaneous ventricular arrhythmias, including polymorphic ventricular tachycardia and ventricular fibrillation. Isolated cardiomyocytes demonstrated prolonged action potential duration caused by reduced expression and function of the potassium channels responsible for repolarization. Short-term exposure to pioglitazone, a PPAR&ggr; agonist, had no effect on mortality or rhythm in WT mice but further exacerbated the arrhythmic phenotype and increased the mortality in the PPAR&ggr; transgenic mice. Conclusions— Our findings support an important link between PPAR&ggr; activation, cardiomyocyte lipid accumulation, ion channel remodeling, and increased cardiac mortality.Background Diabetes and obesity, which confer an increased risk of sudden cardiac death, are associated with cardiomyocyte lipid accumulation and altered cardiac electrical properties, manifested by prolongation of the QRS duration and QT interval. It is difficult to distinguish the contribution of cardiomyocyte lipid accumulation versus the contribution of global metabolic defects to the increased incidence of sudden death and electrical abnormalities.

Mice With Cardiac Overexpression of Peroxisome Proliferator–Activated Receptor γ Have Impaired Repolarization and Spontaneous Fatal Ventricular ArrhythmiasClinical Perspective

Background— Diabetes mellitus and obesity, which confer an increased risk of sudden cardiac death, are associated with cardiomyocyte lipid accumulation and altered cardiac electric properties, manifested by prolongation of the QRS duration and QT interval. It is difficult to distinguish the contribution of cardiomyocyte lipid accumulation from the contribution of global metabolic defects to the increased incidence of sudden death and electric abnormalities. Methods and Results— In order to study the effects of metabolic abnormalities on arrhythmias without the complex systemic effects of diabetes mellitus and obesity, we studied transgenic mice with cardiac-specific overexpression of peroxisome proliferator–activated receptor &ggr; 1 (PPAR&ggr;1) via the cardiac &agr;-myosin heavy-chain promoter. The PPAR&ggr; transgenic mice develop abnormal accumulation of intracellular lipids and die as young adults before any significant reduction in systolic function. Using implantable ECG telemeters, we found that these mice have prolongation of the QRS and QT intervals and spontaneous ventricular arrhythmias, including polymorphic ventricular tachycardia and ventricular fibrillation. Isolated cardiomyocytes demonstrated prolonged action potential duration caused by reduced expression and function of the potassium channels responsible for repolarization. Short-term exposure to pioglitazone, a PPAR&ggr; agonist, had no effect on mortality or rhythm in WT mice but further exacerbated the arrhythmic phenotype and increased the mortality in the PPAR&ggr; transgenic mice. Conclusions— Our findings support an important link between PPAR&ggr; activation, cardiomyocyte lipid accumulation, ion channel remodeling, and increased cardiac mortality.Background Diabetes and obesity, which confer an increased risk of sudden cardiac death, are associated with cardiomyocyte lipid accumulation and altered cardiac electrical properties, manifested by prolongation of the QRS duration and QT interval. It is difficult to distinguish the contribution of cardiomyocyte lipid accumulation versus the contribution of global metabolic defects to the increased incidence of sudden death and electrical abnormalities.