Dynamic reciprocity of sodium and potassium channel expression in a macromolecular complex controls cardiac excitability and arrhythmia.

The cardiac electrical impulse depends on an orchestrated interplay of transmembrane ionic currents in myocardial cells. Two critical ionic current mechanisms are the inwardly rectifying potassium current (I(K1)), which is important for maintenance of the cell resting membrane potential, and the sodium current (I(Na)), which provides a rapid depolarizing current during the upstroke of the action potential. By controlling the resting membrane potential, I(K1) modifies sodium channel availability and therefore, cell excitability, action potential duration, and velocity of impulse propagation. Additionally, I(K1)-I(Na) interactions are key determinants of electrical rotor frequency responsible for abnormal, often lethal, cardiac reentrant activity. Here, we have used a multidisciplinary approach based on molecular and biochemical techniques, acute gene transfer or silencing, and electrophysiology to show that I(K1)-I(Na) interactions involve a reciprocal modulation of expression of their respective channel proteins (Kir2.1 and Na(V)1.5) within a macromolecular complex. Thus, an increase in functional expression of one channel reciprocally modulates the other to enhance cardiac excitability. The modulation is model-independent; it is demonstrable in myocytes isolated from mouse and rat hearts and with transgenic and adenoviral-mediated overexpression/silencing. We also show that the post synaptic density, discs large, and zonula occludens-1 (PDZ) domain protein SAP97 is a component of this macromolecular complex. We show that the interplay between Na(v)1.5 and Kir2.1 has electrophysiological consequences on the myocardium and that SAP97 may affect the integrity of this complex or the nature of Na(v)1.5-Kir2.1 interactions. The reciprocal modulation between Na(v)1.5 and Kir2.1 and the respective ionic currents should be important in the ability of the heart to undergo self-sustaining cardiac rhythm disturbances.

Loss of H3K4 methylation destabilizes gene expression patterns and physiological functions in adult murine cardiomyocytes.

Histone H3 lysine 4 (H3K4me) methyltransferases and their cofactors are essential for embryonic development and the establishment of gene expression patterns in a cell-specific and heritable manner. However, the importance of such epigenetic marks in maintaining gene expression in adults and in initiating human disease is unclear. Here, we addressed this question using a mouse model in which we could inducibly ablate PAX interacting (with transcription-activation domain) protein 1 (PTIP), a key component of the H3K4me complex, in cardiac cells. Reducing H3K4me3 marks in differentiated cardiomyocytes was sufficient to alter gene expression profiles. One gene regulated by H3K4me3 was Kv channel-interacting protein 2 (Kcnip2), which regulates a cardiac repolarization current that is downregulated in heart failure and functions in arrhythmogenesis. This regulation led to a decreased sodium current and action potential upstroke velocity and significantly prolonged action potential duration (APD). The prolonged APD augmented intracellular calcium and in vivo systolic heart function. Treatment with isoproterenol and caffeine in this mouse model resulted in the generation of premature ventricular beats, a harbinger of lethal ventricular arrhythmias. These results suggest that the maintenance of H3K4me3 marks is necessary for the stability of a transcriptional program in differentiated cells and point to an essential function for H3K4me3 epigenetic marks in cellular homeostasis.

Structural heterogeneity promotes triggered activity, reflection and arrhythmogenesis in cardiomyocyte monolayers.

Patients with structural heart disease are predisposed to arrhythmias by incompletely understood mechanisms. We hypothesized that tissue expansions promote source-to-sink mismatch leading to early after-depolarizations (EADs) and reflection of impulses in monolayers of well-polarized neonatal rat ventricular cardiomyocytes.We traced electrical propagation optically in patterned monolayers consisting of two wide regions connected by a thin isthmus.Structural heterogeneities provided a substrate for EADs, retrograde propagation along the same pathway (reflection) and reentry initiation. Reflection always originated during the action potential (AP) plateau at the distal expansion. To determine whether increased sodium current(INa) would promote EADs, we employed adenoviral transfer of Nav1.5 (Ad-Nav1.5). Compared with uninfected and adenoviral expression of green fluorescent protein (Ad-GFP; viral control),Ad-Nav1.5 significantly increased Nav1.5 protein expression, peak and persistent INa density, A Pupstroke velocity, AP duration, conduction velocity and EAD incidence, as well as reflection incidence (29.2%, n =48 vs. uninfected, 9.4%, n =64; and Ad-GFP, 4.8%, n =21). Likewise,the persistent INa agonist veratridine (0.05­3 µM) prolonged the AP, leading to EADs and reflection. Reflection led to functional reentry distally and bigeminal and trigeminal rhythms proximally. Reflection was rare in the absence of structural heterogeneities.Computer simulations demonstrated the importance of persistent INa in triggering reflection and predicted that the gradient between the depolarizing cells at the distal expansion and the repolarizing cells within the isthmus enabled retrograde flow of depolarizing electrotonic current to trigger EADs and reflection. A combination of a substrate (structural heterogeneity) and a trigger (increased persistent INa and EADs) promotes reflection and arrhythmogenesis.

Increased tyrosine phosphorylation mediates the cooling-induced contraction and increased vascular reactivity of Raynaud's disease.

OBJECTIVE: Increased levels of protein tyrosine kinase (PTK) are mechanistically associated with increased contractile responsiveness to cooling. This study tests the hypothesis that increased PTK activity mediates the increased vascular reactivity to agonists and cooling associated with primary Raynaud's disease (RD). METHODS: The response of dermal arterioles isolated from control (n = 29) and RD (n = 29) subjects to contractile and dilatory agents at 37 degrees C and 31 degrees C was characterized using the microvessel perfusion technique. Fluorescence immunohistochemistry was used to measure tyrosine phosphorylation. RESULTS: At 37 degrees C, arteries from RD patients exhibited similar sensitivity to the specific alpha(2)-adrenergic agonist UK 14,304, to serotonin, and to angiotensin II. At 31 degrees C, however, the response to all 3 agonists was greater in the arterioles from the RD patients than in those from the control subjects. Agonist-induced contraction at both temperatures was reversed by cumulative addition of the PTK inhibitors genistein (1-30 microM) and tyrphostin 47 (0.1-10 microM). All arterioles from control subjects relaxed slightly in response to cooling, whereas more than half of those from RD patients contracted. This cooling-induced contraction was reversed by the cumulative addition of genistein. The 3 agonists elicited large increases in tyrosine phosphorylation only in arterial segments from RD patients at 31 degrees C. Cooling from 37 degrees C to 31 degrees C elicited a large increase in tyrosine phosphorylation in arterioles from RD patients, but not those from control subjects. All increases in tyrosine phosphorylation could be prevented by genistein. CONCLUSION: Increased tyrosine phosphorylation mediates cooling-induced contraction and the increased vascular reactivity of skin arterioles from individuals with RD.