RESUMO
Cardiac arrhythmias are associated with various forms of heart diseases. Ventricular arrhythmias present a significant risk for sudden cardiac death. Atrial fibrillations predispose to blood clots leading to stroke and heart attack. Scientists have been developing patch-clamp technology to study ion channels and action potentials (APs) underlying cardiac excitation and arrhythmias. Beyond the traditional patch-clamp techniques, innovative new techniques were developed for studying complex arrhythmia mechanisms. Here we review the recent development of methods including AP-Clamp, Dynamic Clamp, AP-Clamp Sequential Dissection, and Patch-Clamp-in-Gel. These methods provide powerful tools for researchers to decipher how the dynamic systems in excitation-Ca2+ signaling-contraction feedforward and feedback to control cardiac function and how their dysregulations lead to heart diseases.
Las arritmias cardiacas están asociadas a diferentes tipos de enfermedad cardiaca. Las arritmias ventriculares constituyen un alto riesgo de muerte súbita. La fibrilación auricular predispone a coágulos sanguíneos que pueden producir accidentes cerebrovasculares e infarto miocárdico. Los científicos han desarrollado la técnica de patch-clamp para estudiar los canales iónicos y los potenciales de acción (PAs), que constituyen la base de la excitación y las arritmias cardiacas. Además de las clásicas técnicas de patch-clamp, se desarrollaron técnicas innovativas para estudiar los mecanismos complejos de las arritmias. En este trabajo, describimos diferentes métodos recientemente desarrollados tales como AP-clamp ("clampeo" del PA), Dynamic Clamp ("clampeo" dinámico), AP-Clamp Sequential Dissection, (disección secuencial del "clampeo" del AP), y Patch-Clamp-in-Gel (Patch clamp en gel). Estos métodos constituyen herramientas poderosas para descifrar cómo los sistemas dinámicos que constituyen la excitación-las señales de Ca2+ y la contracción, se retroalimentan para controlar la función cardiaca y cómo sus alteraciones llevan a la enfermedad cardiaca.
RESUMO
ß-adrenergic (ß-AR) signaling is essential for the adaptation of the heart to exercise and stress. Chronic stress leads to the activation of Ca2+/calmodulin-dependent kinase II (CaMKII) and protein kinase D (PKD). Unlike CaMKII, the effects of PKD on excitation-contraction coupling (ECC) remain unclear. To elucidate the mechanisms of PKD-dependent ECC regulation, we used hearts from cardiac-specific PKD1 knockout (PKD1 cKO) mice and wild-type (WT) littermates. We measured calcium transients (CaT), Ca2+ sparks, contraction and L-type Ca2+ current in paced cardiomyocytes under acute ß-AR stimulation with isoproterenol (ISO; 100 nM). Sarcoplasmic reticulum (SR) Ca2+ load was assessed by rapid caffeine (10 mM) induced Ca2+ release. Expression and phosphorylation of ECC proteins phospholambam (PLB), troponin I (TnI), ryanodine receptor (RyR), sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) were evaluated by western blotting. At baseline, CaT amplitude and decay tau, Ca2+ spark frequency, SR Ca2+ load, L-type Ca2+ current, contractility, and expression and phosphorylation of ECC protein were all similar in PKD1 cKO vs. WT. However, PKD1 cKO cardiomyocytes presented a diminished ISO response vs. WT with less increase in CaT amplitude, slower [Ca2+]i decline, lower Ca2+ spark rate and lower RyR phosphorylation, but with similar SR Ca2+ load, L-type Ca2+ current, contraction and phosphorylation of PLB and TnI. We infer that the presence of PKD1 allows full cardiomyocyte ß-adrenergic responsiveness by allowing optimal enhancement in SR Ca2+ uptake and RyR sensitivity, but not altering L-type Ca2+ current, TnI phosphorylation or contractile response. Further studies are necessary to elucidate the specific mechanisms by which PKD1 is regulating RyR sensitivity. We conclude that the presence of basal PKD1 activity in cardiac ventricular myocytes contributes to normal ß-adrenergic responses in Ca2+ handling.