Defects in T-Tubular Electrical Activity Underlie Local Alterations of Calcium Release in Heart Failure
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Defects in T-tubular electrical activity underlie local alterations of calcium release in heart failure Claudia Crocinia, Raffaele Coppinib, Cecilia Ferrantinic, Ping Yand, Leslie M. Loewd, Chiara Tesic, Elisabetta Cerbaib, Corrado Poggesic, Francesco S. Pavonea,e,f, and Leonardo Sacconia,f,1 aEuropean Laboratory for Non-Linear Spectroscopy, 50019 Florence, Italy; bDivision of Pharmacology, Department “NeuroFarBa,” University of Florence, 50139 Florence, Italy; cDivision of Physiology, Department of Experimental and Clinical Medicine, University of Florence, 50134 Florence, Italy; dR. D. Berlin Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, CT 06030; eDepartment of Physics and Astronomy, University of Florence, 50019 Sesto Fiorentino, Italy; and fNational Institute of Optics, National Research Council, 50125 Florence, Italy Edited by Clara Franzini-Armstrong, University of Pennsylvania Medical Center, Philadelphia, PA, and approved September 15, 2014 (received for review June 20, 2014) Action potentials (APs), via the transverse axial tubular system in a rat model of postischemic HF, structurally remodeled TATS + (TATS), synchronously trigger uniform Ca2 release throughout the exhibits abnormal electrical activity, i.e., failure of AP propagation cardiomyocyte. In heart failure (HF), TATS structural remodeling and presence of local spontaneous depolarizations. Tubular AP occurs, leading to asynchronous Ca2+ release across the myocyte failures and spontaneous activity can potentially aggravate asyn- + and contributing to contractile dysfunction. In cardiomyocytes from chronous Ca2 release and determine nonhomogeneous myofibril + failing rat hearts, we previously documented the presence of TATS contraction. Simultaneous recording of local Ca2 release and AP in elements which failed to propagate AP and displayed spontaneous the tubular network is needed to unravel the consequences of these + + electrical activity; the consequence for Ca2 release remained, how- electrical anomalies on intracellular Ca2 dynamics. To address this ever, unsolved. Here, we develop an imaging method to simulta- challenge, here we augment the previous experimental setup by + + neously assess TATS electrical activity and local Ca2 release. In HF adding the capability to optically measure Ca2 transients si- cardiomyocytes, sites where T-tubules fail to conduct AP show multaneously with AP in several tubular elements. We apply 2+ a slower and reduced local Ca transient compared with regions this method to dissect the spatiotemporal relationship between + with electrically coupled elements. It is concluded that TATS electrical TATS electrical activity and Ca2 release in heart failure. remodeling is a major determinant of altered kinetics, amplitude, and homogeneity of Ca2+ release in HF. Moreover, spontaneous de- Results polarization events occurring in failing T-tubules can trigger local Isolated rat cardiomyocytes are stained with FluoForte GFP- 2+ 2+ + Ca release, resulting in Ca sparks. The occurrence of tubule- certified, a Ca2 indicator, and di-4-AN(F)EPPTEA, a fluori- 2+ driven depolarizations and Ca sparks may contribute to the ar- nated VSD (9). Fig. 1 illustrates the simultaneous measurement + rhythmic burden in heart failure. of AP and Ca2 transients in an isolated cardiomyocyte. A ran- dom access multiphoton (RAMP) microscope (Fig. 1A) is used cardiac disease | voltage imaging | calcium imaging | nonlinear microscopy to simultaneously excite both dyes, but differently from our previous work; here band-pass filters are used to select the two he simultaneous and coherent recruitment of cardiomyocytes distinct spectral ranges of the fluorescence emission spectra. A Tthat occurs at every heartbeat is fundamental to guarantee spectral unmixing procedure is applied to properly uncouple the + a proper and healthy contraction of the whole heart. Moreover, Ca2 and the voltage signals (Fig. S1). Fig. 1B shows a specific mammalian ventricular cardiomyocytes are provided with a com- plex network of sarcolemmal invaginations called the transverse- Significance axial tubular system (TATS) (1, 2). TATS allows the action po- tential (AP) to propagate rapidly into the cardiomyocyte core. + The plasma membrane of cardiac myocytes contains complex During the AP, Ca2 enters the cell through depolarization- 2+ invaginations known as transverse tubules (T-tubules). In heart activated Ca channels (dihydropyridine receptors, DHPRs) and 2+ 2+ failure, T-tubule loss is a major contributor to Ca transient ab- triggers Ca release (calcium-induced calcium release, CICR) from normalities, leading to weaker and slower contraction. Current the sarcoplasmic reticulum (SR) through the ryanodine receptor 2 2+ 2+ therapeutic strategies are often based on attempts to accelerate (RyR2). The free intracellular Ca concentration ([Ca ]i)rises 2+ 2+ Ca transients. Here, we demonstrate that T-tubular loss repre- and Ca binds to troponin C (TnC), leading to myofilament acti- sents just one way by which T-tubule dysfunction leads to asyn- + vation and contraction. The well-organized topographical extension chronous Ca2 release across the myocyte. In fact, we report that of T-tubules along each sarcomere Z-line profile ensures a homo- 2+ 2+ defects in T-tubular electrical activity may contribute to Ca - geneous Ca release and, consequently, a synchronous contraction mediated arrhythmogenesis not only by favoring asynchronous across the whole cardiomyocyte (3). Ca2+ release, but also by generating voltage-associated Ca2+ Structural alterations and loss of T-tubules have been found in sparks. This work provides the first description to our knowledge several human pathological conditions, including chronic heart of these novel proarrhythmogenic events that could help guide failure (HF) (4, 5). HF is characterized by weakened heart con- future therapeutic strategies. traction, which leads to maladaptive remodeling, further weakening cardiac contraction and potentially causing deadly arrhythmias (6). Author contributions: C.C., R.C., C.F., C.T., E.C., C.P., F.S.P., and L.S. designed research; C.C., Loss and disorganization of the TATS are early features of car- R.C., C.F., and L.S. performed research; P.Y. and L.M.L. contributed new reagents/analytic diomyocyte remodeling in HF, leading to orphaned RyR2 channels tools; C.C. and L.S. analyzed data; and C.C., R.C., C.F., L.M.L., C.T., E.C., C.P., F.S.P., and L.S. + and thus determining nonhomogeneous Ca2 release (7, 8). Re- wrote the paper. cently, using random access microscopy in combination with fluo- The authors declare no conflict of interest. rinated voltage-sensitive dyes (VSD) (9), we have probed the This article is a PNAS Direct Submission. electrical activity of multiple TATS elements within isolated car- Freely available online through the PNAS open access option. diomyocytes, highlighting that the presence of a tight electrical 1To whom correspondence should be addressed. Email: [email protected]. coupling between T-tubular system and surface sarcolemma is en- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sured only by intact TATS (10). In fact, we have demonstrated that 1073/pnas.1411557111/-/DCSupplemental. 15196–15201 | PNAS | October 21, 2014 | vol. 111 | no. 42 www.pnas.org/cgi/doi/10.1073/pnas.1411557111 Downloaded by guest on September 23, 2021 scanned line (5–10 CRUs in ∼10 μm3) (11, 12). The recruitment of CRUs probed on each scanned line may vary stochastically, leading to beat-to-beat changes of the recorded transients. Iso- proterenol increases the open probability of the CRUs, enhanc- + ing Ca2 transients synchronicity. The ability of our method to probe the spatiotemporal re- + lationship between Ca2 and electrical activity is then explored in a model of acute detubulation. Using a formamide-based osmotic-shock technique, it is possible to physically disconnect tubules from the SS (10, 13). Disconnecting TTs would prevent the diffusion of the dye into TATS. However, if staining is per- formed before detubulation, even SS-disconnected TTs will maintain their labeling (Fig. 3A). As previously demonstrated (10), in this condition we find that the elicited AP is clearly visible in the SS, but it is absent in (74 ± 6)% of TTs. Fig. 3B depicts three representative traces (average of 10 trials) from the cell shown in Fig. 3A (white lines). SS, as well as TT1, exhibits 2+ a proper AP and Ca transient; TT2, on the other hand, fails to propagate AP, indicating it underwent the formamide-induced + disconnection. Our method of simultaneous AP and Ca2 re- 2+ A cording highlights an interesting future of disconnecting tubules: Fig. 1. Simultaneous multisite voltage and Ca recording. ( ) Scheme of 2+ the random access multiphoton (RAMP) microscope. It consists of a 1064-nm TT2 shows a remarkably delayed Ca transient. Superimposing 2+ fiber laser, an acousto-optic modulator (AOM) for angular-spreading pre- alternatively TT1 or TT2 on SS Ca traces, it is clear that TT1 2+ 2+ compensation, and two orthogonally mounted acousto-optic deflectors Ca transient is comparable to that at SS, whereas TT2 Ca (AODs) (AOD-x and AOD-y) for laser scanning. The fluorescence signal is transient is delayed (Fig. 3C). A close-up of the TT2 and SS + collected in forward and backward directions using four independent pho- superimposition shows that such delay mainly affects Ca2 rise, tomultipliers (PMTs), two for the voltage and two for the calcium signals. 2+