isolation-functional-characterization-human-ventricular

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

University of Florence

The plasma membrane of cardiac myocytes presents complex invaginations known as the transverse-axial tubular system (TATS). Despite TATS's crucial role in excitation-contraction coupling and morphological alterations found in pathological settings, TATS's electrical activity has never been directly investigated in remodeled tubular networks. Here we develop an ultrafast random access multiphoton microscope that, in combination with a customly synthesized voltage-sensitive dye, is used to simultaneously measure action potentials (APs) at multiple sites within the sarcolemma with submillisecond temporal and submicrometer spatial resolution in real time. We find that the tight electrical coupling between different sarcolemmal domains is guaranteed only within an intact tubular system. In fact, acute detachment by osmotic shock of most tubules from the surface sarcolemma prevents AP propagation not only in the disconnected tubules, but also in some of the tubules that remain connected with the surface. This indicates that a structural disorganization of the tubular system worsens the electrical coupling between the TATS and the surface. The pathological implications of this finding are investigated in failing hearts. We find that AP propagation into the pathologically remodeled TATS frequently fails and may be followed by local spontaneous electrical activity. Our findings provide insight on the relationship between abnormal TATS and asynchronous calcium release, a major determinant of cardiac contractile dysfunction and arrhythmias.

Action potential propagation in transverse-axial tubular system is impaired in heart failure.

The management of patients with hypertrophic cardiomyopathy (HCM) and refractory symptoms due to massive hypertrophy and severe diastolic dysfunction represents a real challenge for the clinical cardiologist. Such patients often require novel therapeutic approaches, both invasive and pharmacological, involving multidisciplinary teamwork; however, the implementation of potentially viable treatment options is hindered by lack of disease-specific evidence. We report the case of a young woman with severe HCM and restrictive physiology, who underwent extensive myectomy via the transaortic and transapical approach, followed by biventricular pacing for cardiac resynchronization, with significant but incomplete symptomatic improvement. The subsequent introduction of ranolazine, based on promising preclinical data, has led to an excellent final result. An ongoing randomized clinical trial is currently testing the efficacy of ranolazine in symptomatic HCM.

[Management of refractory symptoms in hypertrophic cardiomyopathy with restrictive pathophysiology: novel perspectives for ranolazine].

Hypertrophic cardiomyopathy (HCM), the most common mendelian heart disorder, remains an orphan of disease-specific pharmacological treatment because of the limited understanding of cellular mechanisms underlying arrhythmogenicity and diastolic dysfunction.

Late sodium current inhibition reverses electromechanical dysfunction in human hypertrophic cardiomyopathy.

In the developing and mature central nervous system, NG2 expressing cells comprise a population of cycling oligodendrocyte progenitor cells (OPCs) that differentiate into mature, myelinating oligodendrocytes (OLGs). OPCs are also characterized by high motility and respond to injury by migrating into the lesioned area to support remyelination. K(+) currents in OPCs are developmentally regulated during differentiation. However, the mechanisms regulating these currents at different stages of oligodendrocyte lineage are poorly understood. Here we show that, in cultured primary OPCs, the purinergic G-protein coupled receptor GPR17, that has recently emerged as a key player in oligodendrogliogenesis, crucially regulates K(+) currents. Specifically, receptor stimulation by its agonist UDP-glucose enhances delayed rectifier K(+) currents without affecting transient K(+) conductances. This effect was observed in a subpopulation of OPCs and immature pre-OLGs whereas it was absent in mature OLGs, in line with GPR17 expression, that peaks at intermediate phases of oligodendrocyte differentiation and is thereafter downregulated to allow terminal maturation. The effect of UDP-glucose on K(+) currents is concentration-dependent, blocked by the GPR17 antagonists MRS2179 and cangrelor, and sensitive to the K(+) channel blocker tetraethyl-ammonium, which also inhibits oligodendrocyte maturation. We propose that stimulation of K(+) currents is responsible for GPR17-induced oligodendrocyte differentiation. Moreover, we demonstrate, for the first time, that GPR17 activation stimulates OPC migration, suggesting an important role for this receptor after brain injury. Our data indicate that modulation of GPR17 may represent a strategy to potentiate the post-traumatic response of OPCs under demyelinating conditions, such as multiple sclerosis, stroke, and brain trauma.

UDP-glucose enhances outward K(+) currents necessary for cell differentiation and stimulates cell migration by activating the GPR17 receptor in oligodendrocyte precursors.

Poor adherence to medications is a major health concern especially among older subjects. To plan future studies to improve adherence, an epidemiological study, called "Fiesole Misurata", was conducted. The aim of the present paper was to verify the representativeness of the database in evaluating the AntiHyperTensives (AHTs)-taking behaviour.

Representativeness of the "Fiesole Misurata" study database for use in pharmaco-epidemiological investigations on adherence to antihypertensive medications.

Despite the evidence that the hyperpolarization-activated current (If) is highly modulated in human cardiomyopathies, no definite data exist in chronic atrial fibrillation (cAF). We investigated the expression, function, and modulation of If in human cAF.

Chronic atrial fibrillation alters the functional properties of If in the human atrium.

Response to letter regarding article, "Late sodium current inhibition reverses electromechanical dysfunction in human hypertrophic cardiomyopathy".

Early growth response-1 one gene (Egr-1), one of the immediate early response genes, plays an important role in the adaptive response of the myocardium to hypertrophic stimuli. We aimed to investigate the effects of Egr-1 deletion on cardiac function. Egr-1 knock-out (Egr-1(-/-)) homozygous mice were employed to evaluate the electrophysiological and molecular properties of left ventricular cardiomyocytes (VCM) by using patch-clamp technique, intracellular calcium measurements, real-time PCR, and Western blot. Action potential was prolonged and diastolic potential was positive-shifted in VCMs isolated from Egr-1(-/-) mice, in comparison with those from their wild-type (WT) littermates. The calcium content of the sarcoplasmic reticulum was reduced and the decay time for steady-state calcium transient slowed down. Serca2, Ryr, L-type Ca(2+)-channel, and PLB mRNA expression were reduced in Egr-1(-/-) mice compared with the controls. Moreover, Serca2 protein was reduced, while the amount of Ncx1 protein was increased in Egr-1(-/-) hearts compared with those of the WT littermates. Furthermore, genes involved in heart development (GATA-4, TGF-β) and in Egr-1 regulation (Nab1, Nab2) were down regulated in Egr-1(-/-) mice. These results suggest that Egr-1 plays a pivotal role in regulating excitation-contraction coupling in cardiac myocytes.

Altered calcium regulation in isolated cardiomyocytes from Egr-1 knock-out mice.

Transmembrane sodium (Na(+)) fluxes and intracellular sodium homeostasis are central players in the physiology of the cardiac myocyte, since they are crucial for both cell excitability and for the regulation of the intracellular calcium concentration. Furthermore, Na(+) fluxes across the membrane of mitochondria affect the concentration of protons and calcium in the matrix, regulating mitochondrial function. In this review we first analyze the main molecular determinants of sodium fluxes across the sarcolemma and the mitochondrial membrane and describe their role in the physiology of the healthy myocyte. In particular we focus on the interplay between intracellular Ca(2+) and Na(+). A large part of the review is dedicated to discuss the changes of Na(+) fluxes and intracellular Na(+) concentration([Na(+)]i) occurring in cardiac disease; we specifically focus on heart failure and hypertrophic cardiomyopathy, where increased intracellular [Na(+)]i is an established determinant of myocardial dysfunction. We review experimental evidence attributing the increase of [Na(+)]i to either decreased Na(+) efflux (e.g. via the Na(+)/K(+) pump) or increased Na(+) influx into the myocyte (e.g. via Na(+) channels). In particular, we focus on the role of the "late sodium current" (INaL), a sustained component of the fast Na(+) current of cardiac myocytes, which is abnormally enhanced in cardiac diseases and contributes to both electrical and contractile dysfunction. We analyze the pathophysiological role of INaL enhancement in heart failure and hypertrophic cardiomyopathy and the consequences of its pharmacological modulation, highlighting the clinical implications. The central role of Na(+) fluxes and intracellular Na(+) physiology and pathophysiology of cardiac myocytes has been highlighted by a large number of recent works. The possibility of modulating Na(+) inward fluxes and [Na(+)]i with specific INaL inhibitors, such as ranolazine, has made Na(+)a novel suitable target for cardiac therapy, potentially capable of addressing arrhythmogenesis and diastolic dysfunction in severe conditions such as heart failure and hypertrophic cardiomyopathy.

Article	Year
Action potential propagation in transverse-axial tubular system is impaired in heart failure. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 22451916	2012
[Management of refractory symptoms in hypertrophic cardiomyopathy with restrictive pathophysiology: novel perspectives for ranolazine]. Giornale italiano di cardiologia (2006)\| PubMed ID: 22495647	2012
Late sodium current inhibition reverses electromechanical dysfunction in human hypertrophic cardiomyopathy. Circulation\| PubMed ID: 23271797	2013
UDP-glucose enhances outward K(+) currents necessary for cell differentiation and stimulates cell migration by activating the GPR17 receptor in oligodendrocyte precursors. Glia\| PubMed ID: 23640798	2013
Representativeness of the "Fiesole Misurata" study database for use in pharmaco-epidemiological investigations on adherence to antihypertensive medications. Aging clinical and experimental research\| PubMed ID: 23780690	2013
Chronic atrial fibrillation alters the functional properties of If in the human atrium. Journal of cardiovascular electrophysiology\| PubMed ID: 23869794	2013
Response to letter regarding article, "Late sodium current inhibition reverses electromechanical dysfunction in human hypertrophic cardiomyopathy". Circulation\| PubMed ID: 24002721	2013
Altered calcium regulation in isolated cardiomyocytes from Egr-1 knock-out mice. Canadian journal of physiology and pharmacology\| PubMed ID: 24289086	2013
Regulation of intracellular Na(+) in health and disease: pathophysiological mechanisms and implications for treatment. Global cardiology science & practice\| PubMed ID: 24689024	2013

Raffaele Coppini

Biography

JoVE Journal Publications

Other Publications