Targeted disruption of the voltage-dependent calcium channel alpha2/delta-1-subunit.

Cardiac L-type voltage-dependent Ca(2+) channels are heteromultimeric polypeptide complexes of alpha(1)-, alpha(2)/delta-, and beta-subunits. The alpha(2)/delta-1-subunit possesses a stereoselective, high-affinity binding site for gabapentin, widely used to treat epilepsy and postherpetic neuralgic pain as well as sleep disorders. Mutations in alpha(2)/delta-subunits of voltage-dependent Ca(2+) channels have been associated with different diseases, including epilepsy. Multiple heterologous coexpression systems have been used to study the effects of the deletion of the alpha(2)/delta-1-subunit, but attempts at a conventional knockout animal model have been ineffective. We report the development of a viable conventional knockout mouse using a construct targeting exon 2 of alpha(2)/delta-1. While the deletion of the subunit is not lethal, these animals lack high-affinity gabapentin binding sites and demonstrate a significantly decreased basal myocardial contractility and relaxation and a decreased L-type Ca(2+) current peak current amplitude. This is a novel model for studying the function of the alpha(2)/delta-1-subunit and will be of importance in the development of new pharmacological therapies.

Sarcoplasmic reticulum adenosine triphosphatase overexpression in the L-type Ca2+ channel mouse results in cardiomyopathy and Ca2+ -induced arrhythmogenesis.

BACKGROUND: Overexpression of the L-type voltage-dependent calcium channel alpha(1C)-subunit (L-VDCC OE) in transgenic mice results in adaptive hypertrophy followed by a maladaptive phase associated with a decrease in sarcoplasmic reticulum adenosine triphosphatase (SERCA)2a expression at 8 to 10 months of age. Overexpressing SERCA to manipulate calcium (Ca(2+)) cycling and prevent pathologic phenotypes in some models of heart failure has been proven to be a promising genetic strategy. OBJECTIVE: In this study we investigated whether genetic manipulation that increases Ca(2+) uptake into the sarcoplasmic reticulum by overexpressing SERCA1a (skeletal muscle specific) into the L-VDCC OE background could restore or further deteriorate Ca(2+) cycling, contractile dysfunction, and electrical remodeling in the heart failure phenotype. RESULTS: We found that the survival rate of L-VDCC OE/SERCA1a OE double transgenic mice decreased by 50%. L-VDCC OE/SERCA1a OE mice displayed an accelerated phenotype of severe dilation of both ventricles associated with deteriorated left ventricular function. Voltage clamp experiments revealed enhanced increased inward Ca(2+) current density and decreased the transient outward potassium current. Action potential duration in double transgenic ventricular myocytes was prolonged, and isoproterenol induced early after depolarization. These mice demonstrated a high incidence of spontaneous left ventricular arrhythmia. Expression of the proarrhythmic signaling protein Ca(2+)/calmodulin-dependent kinase II (CaMKII) was increased while connexin43 expression was decreased, defining an important putative mechanism in the electrophysiologic disturbances and mortality. CONCLUSIONS: Despite previous reports of improved cardiac function in heart failure models after SERCA intervention, our results advocate the need to elucidate the involvement of augmented Ca(2+) cycling in arrhythmogenesis.

Presynaptic stimulus-release and postsynaptic compensatory changes in mice lacking the N-type calcium channel α(1B)-subunit.

N-type (Ca(v)2.2) voltage-dependent calcium channels (VDCC) play an important role in presynaptic neurotransmitter release in the autonomic nervous system and may be clinically relevant in the treatment of cardiovascular diseases. The physiological impact of N-type VDCC ablation on cardiac function, stimulus-release coupling and cardiac autonomic regulation was studied using mice deficient in the α(1B) subunit of the N-type channel (N-type-/-).The positive inotropic effect (increase in +dP/dt) secondary to high frequency field stimulation (HFFS), mediated by the sympathetic nervous system, was decreased by 33 ± 12.6% in N-type-/- versus 89 ± 11.4% in Wild-Type (WT)(P<0.01), whereas the negative inotropic response (decrease in +dP/dt) following HFFS in the presence of propranolol, mediated by the parasympathetic nervous system, was similar to that in Wild-type (WT) animals 34 ± 5.0% and 35 ± 5.4%, respectively. There were no changes in the postsynaptic ß-adrenergic responsiveness, ß-adrenoreceptor density or adenylyl cyclase activity. N-type-/- hearts demonstrated an increased contractile response to α(1)-adrenoreceptor (α(1)-ADR) stimulation with 10(-5)M phenylephrine in the presence of the ß-blocker propranolol, which might be attributed to an increased expression of PLCß1. Protein abundance of other signal transducers for α(1) ADR transduction protein was not changed in the N-type-/- hearts. These results suggest that selective impairment of sympathetic inflow does not modulate postsynaptic ß-adrenergic responsiveness, but causes increased functional response to α(1)-adrenergic stimulation.

Regulating RISK: a role for JAK-STAT signaling in postconditioning?

Postconditioning (POC), a novel strategy of cardioprotection against ischemia-reperfusion injury, is clinically attractive because of its therapeutic application at the predictable onset of reperfusion. POC activates several intracellular kinase signaling pathways, including phosphatidylinositol 3-kinase (PI3K)-Akt (RISK). The regulation of POC-induced survival kinase signaling, however, has not been fully characterized. JAK-STAT activation is integral to cardiac ischemic tolerance and may provide upstream regulation of RISK. We hypothesized that POC requires the activation of both JAK-STAT and RISK signaling. Langendorff-perfused mouse hearts were subjected to 30 min of global ischemia and 40 min of reperfusion, with or without POC immediately after ischemia. A separate group of POC hearts was treated with AG 490, a JAK2 inhibitor, Stattic, a specific STAT3 inhibitor, or LY-294002, a PI3K inhibitor, at the onset of reperfusion. Cardiomyocyte-specific STAT3 knockout (KO) hearts were also subjected to non-POC or POC protocols. Myocardial performance (+dP/dt(max), mmHg/s) was assessed throughout each perfusion protocol. Phosphorylated (p-) STAT3 and Akt expression was analyzed by Western immunoblotting. POC enhanced myocardial functional recovery and increased expression of p-STAT3 and p-Akt. JAK-STAT inhibition abrogated POC-induced functional protection. STAT3 inhibition decreased expression of both p-STAT3 and p-Akt. PI3K inhibition also attenuated POC-induced cardioprotection and reduced p-Akt expression but had no effect on STAT3 phosphorylation. Interestingly, STAT3 KO hearts undergoing POC exhibited improved ischemic tolerance compared with KO non-POC hearts. POC induces myocardial functional protection by activating the RISK pathway. JAK-STAT signaling, however, is insufficient for effective POC without PI3K-Akt activation.