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Summary

A set of protocols are presented that describe the measurement of contractile function via sarcomere length detection along with calcium (Ca²⁺) transient measurement in isolated rat myocytes. The application of this approach for studies in animal models of heart failure is also included.

Abstract

Contractile dysfunction and Ca²⁺ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury and/or remodeling. One approach for assessing these functional alterations utilizes unloaded shortening and Ca²⁺ transient analyses in primary adult cardiac myocytes. For this approach, adult myocytes are isolated by collagenase digestion, made Ca²⁺ tolerant, and then adhered to laminin-coated coverslips, followed by electrical pacing in serum-free media. The general protocol utilizes adult rat cardiac myocytes but can be readily adjusted for primary myocytes from other species. Functional alterations in myocytes from injured hearts can be compared to sham myocytes and/or to in vitro therapeutic treatments. The methodology includes the essential elements needed for myocyte pacing, along with the cell chamber and platform components. The detailed protocol for this approach incorporates the steps for measuring unloaded shortening by sarcomere length detection and cellular Ca²⁺ transients measured with the ratiometric indicator Fura-2 AM, as well as for raw data analysis.

Introduction

The analysis of cardiac pump function often requires a range of approaches to gain adequate insight, especially for animal models of heart failure (HF). Echocardiography or hemodynamic measurements provide insight into in vivo cardiac dysfunction¹, while in vitro approaches are often employed to identify whether dysfunction arises from changes in the myofilament and/or the Ca²⁺ transient responsible for coupling excitation, or the action potential, with contractile function (e.g., excitation-contraction [E-C] coupling). In vitro approaches also provide an opportunity to screen the functional response to neurohormones, vector-induced genetic alterations, as well as potential therapeutic agents² prior to pursuing costly and/or laborious in vivo treatment strategies.

Several approaches are available to investigate in vitro contractile function, including force measurements in intact trabeculae³ or permeabilized myocytes⁴, as well as unloaded shortening and Ca²⁺ transients in intact myocytes in the presence and absence of HF^5,6. Each of these approaches focuses on cardiac myocyte contractile function, which is directly responsible for cardiac pump function^2,7. However, the analysis of both contraction and E-C coupling together is most often performed by measuring shortening of the muscle length and Ca²⁺ transients in isolated, Ca²⁺ tolerant adult myocytes. The laboratory utilizes a detailed published protocol to isolate myocytes from rat hearts for this step⁸.

Both the Ca²⁺ transient and myofilaments contribute to shortening and re-lengthening in intact myocytes and can contribute to contractile dysfunction^2,7. Thus, this approach is recommended when in vitro functional analysis requires an intact myocyte containing the Ca²⁺ cycling machinery plus the myofilaments. For example, intact isolated myocytes are desirable for studying contractile function after modifying the myofilament or Ca²⁺ cycling function via gene transfer⁹. In addition, an intact myocyte approach is suggested for analyzing the functional impact of neurohormones when studying the impact of downstream second messenger signaling pathways and/or response to therapeutic agents². An alternative measurement of load-dependent force in single myocytes is most often performed after membrane permeabilization (or skinning) at low temperatures (≤15 °C) to remove the Ca²⁺ transient contribution and focus on myofilament function¹⁰. The measurement of load-dependent force plus Ca²⁺ transients in intact myocytes is rare due largely to the complex and technical challenge of the approach¹¹, especially when higher throughput is needed, such as for measuring responses to neurohormone signaling or as a screen for therapeutic agents. The analysis of cardiac trabeculae overcomes these technical challenges but also may be influenced by non-myocytes, fibrosis, and/or extracellular matrix remodeling². Each of the approaches described above requires a preparation containing adult myocytes because neonatal myocytes and myocytes derived from inducible pluripotent stem cells (iPSCs) do not yet express the full complement of adult myofilament proteins and usually lack the level of myofilament organization present in the adult rod-shaped myocyte². To date, evidence in iPSCs indicates that the full transition to adult isoforms exceeds more than 134 days in culture¹².

Given the focus of this collection on HF, the protocols include approaches and analysis to differentiate contractile function in failing versus non-failing intact myocytes. Representative examples are provided from rat myocytes studied 18-20 weeks after a supra-renal coarctation, described earlier^5,13. Comparisons are then made to myocytes from sham-treated rats.

The protocol and imaging platform described here are used to analyze and monitor changes in shortening and Ca²⁺ transients in rod-shaped cardiac myocytes during the development of HF. For this analysis, 2 x 10⁴ Ca²⁺-tolerant, rod-shaped myocytes are plated on 22 mm² laminin-coated glass coverslips (CSs) and cultured overnight, as described earlier⁸. The components assembled for this imaging platform, along with the media and buffers used for optimal imaging, are provided in the Table of Materials. A guide for data analysis using a software and the representative results are also provided here. The overall protocol is broken down into separate sub-sections, with the first three sections focusing on isolated rat myocytes and data analysis, followed by cellular Ca²⁺ transient experiments and data analysis in myocytes.

Protocol

Studies performed on rodents followed the Public Health Service Policy on Humane Care and Use of Laboratory Animals and were approved by the University of Michigan Institutional Animal Care and Use Committee. For this study, myocytes were isolated from 3-34-month-old Sprague-Dawley and F344BN rats weighing ≥ 200 g⁵. Both male and female rates were used.

1. Myocyte pacing for contractile function studies

1. Make fresh M199-based media for each set of isolated myocytes (Table of Materials). 1 day before pacing, plate 2 x 10⁴ Ca²⁺-tolerant, rod-shaped myocytes on 22 mm² laminin-coated glass coverslips (CSs), as described earlier⁸.
2. To prepare the chambers, soak each chamber in 10% bleach for 1 h. Then, flush the chambers with running tap water for 30-45 min, followed by distilled water replaced every 5 min for 30 min, followed by deionized water replaced every 5 min for 30 min, and a final rinse in deionized water.
3. Dry the chambers on a clean surface for 20-30 min (Supplemental Figure 1A-C). Next, UV treat the chambers in a biosafety cabinet for 5 min in each direction (total = 20 min). This step is not needed for pre-sterilized chambers.
   CAUTION: Leave the area to minimize UV exposure.
4. Remove the cover from the chamber, add 3 mL of M199-based media (see Table of Materials) to each well (Supplemental Figure 1B), replace the cover, and preheat the chamber in a 37 °C incubator with 5% CO₂ and 20% O₂.
5. Sterilize forceps in a hot bead sterilizer at 250 °C for 20-25 s. Transfer the preheated stimulation chamber from the incubator to a biosafety cabinet.
6. Transfer each coverslip (CS) containing cardiac myocytes to individual wells in the stimulation chamber using sterile forceps. Transfer CSs four at a time, followed by heating for 10 min prior to the transfer of the remaining four CSs to maintain the media temperature.
7. Attach banana jacks to the stimulation chamber on one end (Supplemental Figure 1C) and to the stimulator on the other end (Supplemental Figure 1D).
8. Set the stimulator frequency to 0.2 Hz and set the voltage (~12 V) to achieve contraction in ~10%-20% of all the rod-shaped cardiac myocytes within a well. To determine the number of contracting myocytes, place the chamber under an inverted microscope with 4x to 10x magnification.
9. Place the stimulation chamber in an incubator at 37 °C in 5% CO₂ (Supplemental Figure 1E). Change the media every 12 h using media preheated in a 37 °C, 5% CO₂ incubator for 20-25 min. Continue electrical stimulation for up to 3 days with the media changed every 12 h.

2. Contractile function analysis of adult rat cardiac myocytes

1. Preheat a gel pack and media in a 37 °C incubator for 30 min prior to the experiment.
2. Turn on the contractile function platform components listed in the Table of Materials and shown in Supplemental Figure 2, with special attention to the key components, which include an anti-vibration table (Supplemental Figure 2A-1) with an inverted microscope with a deep red (590 nm) filter (Supplemental Figure 2A-2,3) and CCD camera (Supplemental Figure 2A-4) connected to a CCD controller (Supplemental Figure 2A-5 and Supplemental Figure 2C-5) and integrated with a PC computer (Supplemental Figure 2B-14).
3. Assemble tubing into the peristaltic pump (Supplemental Figure 2A-8; Supplemental Figure 2A-8), transfer the preheated gel pack into the tube holder (Supplemental Figure 2A-9), and turn on the vacuum (Supplemental Figure 2A-11) and power to the cell stimulator (Supplemental Figure 2B-13).
4. Place a 50 mL tube with media in the insulated tube holder (Supplemental Figure 2A-9) and begin perfusion at ~0.5 mL/min through the peristaltic pump tubing (Supplemental Figure 2E-8).
5. Add a small amount of preheated media to a small weigh boat (~2 mL). Transfer one CS containing myocytes from the stimulation chamber to the weigh boat. Return the stimulation chamber to 37 °C in the 5% CO₂ incubator.
6. Remove the CS from the weigh boat and gently wipe the underside. Transfer the CS to a freshly greased CS chamber (Supplemental Figure 2A,F-10; black arrow) and gently add a drop of media (~200 μL) onto the CS.
7. Place a platinum electrode mount over the CS (Supplemental Figure 2F; gray arrow) and lay a new CS over the top using forceps. Place a top mount (Supplemental Figure 2F; white arrow) over the CS sandwich and then install two or four #0 pan-head screws to finish assembling the chamber.
8. Once the media is present throughout the pump tubing, attach the tubing to the preheater, and then connect the preheater to the CS chamber assembled in step 2.7. Begin perfusion at 0.5 mL/min and place a tissue wipe below the chamber to make sure there are no leaks.
9. Place the CS chamber in the stage adapter. The perfused media is collected at the opposite end of the chamber with tubing connected to a vacuum system. Turn on the heating system (Supplemental Figure 2A,D-7) and equilibrate the chamber, objective, and preheater for ~5-10 min to achieve a constant media temperature of 37 °C. Visualize myocytes with the 40x water immersion objective on the microscope during this equilibration time.
10. Activate the pacing stimulator (Supplemental Figure 2B-13) by setting the voltage to 1.5x the setting needed for 80% of rod-shaped cells to contract, which is typically 35-40 V. Set the stimulation frequency to 0.2 Hz to optimize contractile function and myocyte survival.
11. Collect contractile shortening and re-lengthening traces from continuously perfused and paced myocytes. To collect shortening measurements, use a CCD camera (Supplemental Figure 2A-4) connected to a CCD controller (Supplemental Figure 2B-5,C) and a dedicated computer with an I/O controller board (Supplemental Figure 2B-14; see Table of Materials). The platform measures sarcomere shortening in rat myocytes, although similar results are obtained from myocyte measurements collected from the longitudinal edges of myocytes (see Ecklerle et al.¹⁴).
12. To collect sarcomere shortening traces, open the software on the computer, select OK, and then File > New. Prepare a screen template by selecting Traces > Edit User Limits > Sarcomere Length and then setting maximum and minimum values, which are typically between 2.0 μm and 1.5 μm. Calibrate the CCD camerawith a 0.01 mm graticule prior to making measurements in the myocytes (Figure 1D).
13. To record sarcomere length traces, select File > New. Identify a contracting myocyte and position the myocyte along the longitudinal axis of the camera so the striation pattern is vertical (Figure 1B).
14. Use the computer mouse to place the region of interest (ROI) box (pink box) over the myocyte (Figure 1C). Select Collect and Start to record contractile function. Record shortening for 60 s from each myocyte at low stimulation frequencies (≤0.5 Hz).
15. Record sarcomere shortening from 5-10 cells per CS. Perform measurements at 1 cell/CS if studies include treatment with agonists/antagonists. Prepare separate pre-warmed media and a different, dedicated piece of perfusion tubing loaded in a multi-channel peristaltic pump and follow steps 2.9.-2.14. to measure the impact of drug or neurohormone delivery on myocyte contractile function.
16. To measure shortening over a range of frequencies, pace myocytes at each frequency to obtain steady-state shortening prior to recording⁵. For these studies, double the perfusion rate and begin recording 15-20 s after stimulation at a new frequency to obtain consistent steady-state contractile responses for frequencies in the range of 0.2 Hz and 2 Hz. Typically, record no more than 2 myocytes/CS for shortening across the given range of stimulation frequencies.
17. Record at least 7 contractions/cell to obtain reliable signal-averaged data when analyzing the recordings (see step 3.).

3. Data analysis of contractile function in isolated myocytes

1. To begin, select File, choose a recorded trace, and then select Open. Select Tab 1 (left side) of the base trace and then set the yellow panel at the top of the trace to Sarc-Length. Select Traces and the screen template prepared in step 2.12.
2. To prepare an analysis template, select Operations, Monotonic Transient Analysis Options, TTL Event Mark in the Definition of t0 box, as well as the following options from the menu: Baseline (resting sarcomere length), Departure velocity relative to t0 (dep v), Peak (peak height and %peak height/baseline or bl%peak h), Return velocity relative to tPeak (ret v), Time to Peak 50% (TTP_50%) using t0, and Time to Baseline 50% (TTR_50%) using tPeak. Save these analysis options by selecting Templates > Save Analysis Template with an identifier. Load this analysis template prior to analyzing each trace.
3. To begin data analysis, select Marks > Gate > Add Transient > Convert from Event Mark > Analysis Range. Now set the time range from -0.01 s to 1.20 s for myocytes paced at 0.2 Hz (Figure 2A, red marks on the lower portion of the raw trace). Select a shorter time range for myocytes stimulated at higher frequencies.
4. Produce a signal-averaged trace by selecting Operations > Average Events. A signal-averaged recording appears below the original base trace.
5. Select Tab 1 of the signal-averaged trace (left side of the screen), and then select Marks on the upper menu, followed by Add Transient. Select Operations from the top menu and Monotonic Transient Analysis to display the signal-averaged values for baseline sarcomere length, peak height, bl%peak h, dep v, ret v, TTP_50%, and TTR_50% in the signal-averaged display panel.
6. Transfer each sarcomere transient analysis to a spreadsheet for the composite analysis of multiple myocytes by selecting Export > Monotonic Transient Analysis > Clipboard Current. Paste this data into the spreadsheet for each trace.
7. To copy the signal-averaged trace, select Export > Current Trace > Clipboard > Options and set the decimal places to 5, then select Tabs Delimiter, and click OK, and OK. Paste the signal-averaged traces for each myocyte recording into a second spreadsheet.

4. Recording Ca²⁺ transients in rat adult cardiac myocytes

1. Prior to measuring Ca²⁺ transients, turn on the platform components described in step 2.2., along with additional fluorescence imaging components (see additional components for Ca²⁺ imaging analysis in the Table of Materials; Supplemental Figure 2A-5,6; 2B-12).
2. Prepare Fura-2AM stock solution containing 1 mM Fura-2AM plus 0.5 M probenecid in dimethylsulfoxide (DMSO). Probenecid minimizes Fura-2AM leakage¹⁵.
3. Dilute the stock solution to 5 μM Fura-2AM and 2.5 mM probenecid in 2.5 mL of media (see Table of Materials) in one well of a 6-well plate. Add 2.5 mL media alone (no Fura-2AM) to three additional wells in the plate, cover the plate with aluminum foil, and preheat the media to 37 °C.
4. Transfer a CS containing myocytes from the pacing chamber to the well containing Fura-2AM, cover, and return the plate to the 37 °C incubator with 5% CO₂ for 4 min. Mount tubing in the perfusion pump and perfuse with media during the Fura-2AM loading period, as described in step 2.4.
5. Turn off or minimize room lights to reduce fluorescence photobleaching and to optimize the fluorescence signal. Remove the 6-well plate from the incubator, and then wash the CS for 10 s in each of the three wells containing media alone. Transfer the CS to a small weigh boat containing prewarmed media (no added Fura-2AM).
6. Mount the CS to freshly greased CS chamber after gently wiping the underside of the CS. Gently add a drop of media to the CS, and then assemble the electrode mount over the CS, followed by a second CS and the top mount. Use #0 pan-head screws to finish assembling the cell chamber, as described in steps 2.6.-2.7.
7. Connect the pump tubing filled with media to the preheater, connect the preheater to the CS chamber, and place in a stage adapter, as described in step 2.8. Collect the media with the vacuum tubing system and turn on the heating system (Supplemental Figure 2A,D-7) to equilibrate the chamber to 37°C, as described in steps 2.8.-2.9.
8. Activate the pacing stimulator (Supplemental Figure 2B-13) set to 35-40 V and 0.2 Hz, as described in step 2.10.
9. Prior to recording a Ca²⁺ transient, turn on a small flashlight or booklight mounted near the computer keyboard to help see the keyboard. In addition, record the fluorescence background in an area without cardiac myocytes. To start, select File > Start > New, as described in step 2.12.
10. Select a ROI, and set one tab (or trace bar, left side) for raw numerator and a second tab for raw denominator, defined in the upper-center of each tab. Record the background for 10-20 s. Right-click the mouse and drag over one trace bar panel to obtain the raw signal-averaged numerator and denominator values. Enter these values by selecting the Operations > Constants, and enter the raw values.
11. Prepare a new screen template to collect both shortening and Ca²⁺ transients. For sarcomere length, select Tab 1 and use the same process as described in step 2.13. For Ca²⁺ imaging, select Tab 2 > Ratio (upper-center of tab) > Traces > Edit User Limits to set the minimum and maximum levels for the ratio, which are typically set between 1 and 5. Use the same process to define the numerator and denominator ranges for tabs 3 and 4 (left side).
12. Position a ROI box over a myocyte image, as described in step 2.14. Select File > New > Collect. Now select Start to collect shortening and ratiometric Ca²⁺ data. Record ≥7 contractions/cell for signal-averaged analysis.
13. Repeat the background trace recording described in step 4.10. after recording 2-4 myocytes. Typically, record 4-5 myocytes/CS or fewer myocytes if there are significant changes in the background reading.

5. Data analysis of Ca²⁺ transients in isolated myocytes.

1. Open the desired file for analysis and select Templates > Screen Template for shortening plus Ca²⁺ transients. Next, select tabs 1 and 2 (left) to show sarcomere length and Ca²⁺ ratio, respectively. Select Operations > Constants and enter the numerator and denominator values from the Ca²⁺ background trace.
2. Prepare analysis templates for shortening plus Ca²⁺ transient data. Select tab 1 (left side) and use the same process as described in step 3.2. to set the sarcomere length analysis template.
3. Select tab 2 (left side), and select Operations, Monotonic Transient Analysis Options, TTL event mark in the Definition of t0 box, and the following options from the menu: Baseline (basal ratio), Departure velocity relative to t0 (dep v), Peak (peak height and %peak height/baseline or bl%peak h), Decay velocity relative to tPeak (ret v), Time to Peak 50% (TTP_50%) using t0, and Time to Baseline 50% (TTR_50%) using tPeak. Save these analysis options by selecting Templates and Save Analysis Template using a new identifier name. Load this analysis template prior to analyzing each trace.
4. Use the same process as described in steps 3.3.-3.6. to average the signal and then analyze sarcomere length, followed by the same steps for Ca²⁺ transients. Set separate marks for sarcomere length and for the Ca²⁺ transient ratio trace.

Representative Results

Contractile function studies are performed on rat myocytes starting the day after isolation (day 2) up to 4 days post isolation. Although myocytes can be recorded the day after isolation (i.e., day 2), longer culture times are often required after gene transfer or treatments to modify contractile function⁸. For myocytes cultured for more than 18 h after isolation, the pacing protocol described in section 1 helps maintain t-tubules and consistent shortening and re-lengthening results.

A representative portion of a CS containing myocytes for shortening studies is shown in Figure 1A, along with an appropriately positioned myocyte prior to setting the ROI (Figure 1B). Once a ROI is identified (Figure 1C; pink box), the algorithm information shown below the myocyte also helps to optimize myocyte positioning prior to recording. Specifically, the linear optical density (LOD; black line) is an indicator for the number and spacing of sarcomeres, and a sharp power spectrum in the fast Fourier transform trace (FFT, red line) helps to achieve optimal alignment for the recording of shortening and re-lengthening. The graticule pattern used for the calibration of sarcomere length (and edge detection) is shown in Figure 1D. A typical aligned recording of sarcomere length shortening is shown in Figure 2A (top panel), along with the signal-averaged analysis described in section 3 (lower panel).

Dysfunction is often detected in myocytes when there is in vivo dysfunction in animal models. For example, systolic dysfunction observed by echocardiography in response to pressure overload (PO)¹³ is also detected in myocyte shortening studies⁵. To illustrate data traces, representative raw (Figure 2; upper panel) and signal-averaged (Figure 2; lower panel) traces obtained at 0.2 Hz are shown for myocytes from sham- (Figure 2A) and PO-treated (Figure 2B) rats. To test whether myocyte function could be rescued after PO, viral-mediated gene transfer at the time of myocyte isolation also was used to replace endogenous cardiac troponin I (cTnI) with a phospho-mimetic cTnI T144D substitution (T144D) in the sarcomere¹⁶. The initial analysis shows the PO-induced reduction in contractile function (Table 1, upper panel) returned toward sham levels in PO myocytes 4 days after gene transfer of cTnIT144D (Table 1; lower panel).

This platform also can be used to measure Ca²⁺ transients, along with shortening in isolated myocytes. Shortening and Ca²⁺ were not recorded after PO because PO reduces adherence of rat myocytes to laminin¹³, and a comparable model previously showed altered Ca²⁺ handling develops at a similar time point¹⁷. Instead, representative experiments were performed in Fura-2AM-loaded myocytes isolated from 2-3-month-old rats. A representative recording and signal-averaged traces are shown in Figure 3, along with the data analysis in Table 2. For this set of experiments, myocytes isolated from adult rats were studied 4 days after adenoviral-mediated gene transfer (multiplicity of infection = 100) of cTnIT144D or wild-type cTnI. Both sarcomere shortening and Ca²⁺ transients were measured after loading myocytes with Fura-2AM. Gene transfer of cTnIT144D enhanced peak shortening and elevated diastolic Ca²⁺ levels compared to cTnI in these initial studies (Table 2). While more extensive analysis is needed, the initial results suggest in vivo replacement with cTnIT144D could produce a complex cardiac phenotype due to changes in both contractile function and Ca²⁺ handling over time.

Figure 1: Adult rat cardiac myocytes used for functional studies. (A) Representative isolated cardiac myocytes from an adult rat (scale bar = 50 μm). Arrow points to a representative myocyte imaged for contractile function analysis. (B) Representative myocyte with a ROI (pink) located on the side (scale bar = 20 μm). (C) Representative myocyte with a ROI (upper panel), the sarcomere pattern for this myocyte (lower panel, blue; scale bar = 20 μm), and the sharp peak of the power spectrum (lower panel, red). (D) Screen capture of 0.01 mm graticule to calibrate the shortening measurements. Please click here to view a larger version of this figure.

Figure 2: Representative recordings from rat myocytes. A raw recording (upper panel) and signal-averaged (lower panel) trace recorded at 0.2 Hz in myocytes from (A) sham- and (B) pressure overload (PO)-treated rats. Myocytes were isolated 18-20 weeks after surgery, with PO produced by supra-renal coarctation¹³. Please click here to view a larger version of this figure.

Figure 3: Representative recording and analysis of sarcomere length (SL) and Ca²⁺ transients from Fura-2AM-loaded adult cardiac myocytes. (A) Raw traces for SL, Ca²⁺ transient ratio (ratio), and the numerator and denominator traces used to produce the Ca²⁺ transient ratio. (B) An example of signal-averaged traces for SL (upper trace) and the Ca²⁺ transient ratio (lower trace). (C) Traces are analyzed for sarcomere length (SL) and the Ca²⁺ transient ratio using the monotonic trace algorithm. Please click here to view a larger version of this figure.

Rat group	Sham (n=30)	PO (n=32)
Resting sarcomere length (mm; SL)	1.761 + 0.006	1.748 + 0.004
peak height (% of baseline)	9.003 + 0.409	6.680 + 0.552*
Peak amplitude (mm)	0.159 + 0.007	0.117 + 0.010*
Shortening rate (mm/s)	-4.674 + 0.285	-4.143 + 0.335
Re-lengthening rate (mm/s)	3.251 + 0.223	2.706 + 0.273
Time to peak (ms; TTP)	60 + 2	59 + 3
Time to 50% re-lengthening (ms; TTR50%)	35 + 2	37 + 3
Rat group	Sham + cTnIT144D (n=14)	PO + cTnIT144D (n=17)
Resting sarcomere length (mm; SL)	1.768 + 0.009	1.776 + 0.004
peak height (% of baseline)	9.038 + 1.339	8.414 + 0.960
Peak amplitude (mm)	0.160 + 0.023	0.149 + 0.016
Shortening rate (mm/s)	-5.972 + 0.711	-4.173 + 0.726
Re-lengthening rate (mm/s)	3.925 + 0.577	3.055 + 0.403
Time to peak (ms; TTP)	51 + 5	59 + 2
Time to 50% re-lengthening (ms; TTR50%)	31 + 3	32 + 2

Table 1: Comparison of contractile function in cardiac myocytes in response to pressure overload (PO) and gene transfer. Myocyte shortening results are from sham and PO rat hearts 18-20 weeks post surgery (upper panel)^5,13 and myocytes from sham and PO rats 4 days after cTnIT144D gene transfer (lower panel). Contractile function is measured 4 days after myocyte isolation/gene transfer in all groups. Results are expressed as mean ± SEM (n = number of myocytes). Each set of sham and PO data is compared by a Student's t-test, with *p < 0.05 considered statistically significant. Peak amplitude results for sham and PO myocytes alone were reported earlier in Ravichandran et al.⁵.

	Sarcomere length analysis
Gene transfer group	cTnI (n=21)	cTnIT144D (n=16)
Resting sarcomere length (mm; SL)	1.81 + 0.01	1.81 + 0.01
Peak amplitude (mm)	0.11 + 0.01	0.14 + 0.02*
Shortening rate (mm/s)	-4.29 + 0.42	-4.67 + 0.77
Re-lengthening rate (mm/s)	2.92 + 0.43	3.71 + 0.64
Time to peak (ms; TTP)	50 + 4	84 + 28
Time to 50% re-lengthening (ms; TTR50%)	37 + 4	35 + 6
	Ca2+ transient analysis
Gene transfer group	cTnI (n=21)	cTnIT144D (n=19)
Resting Ca2+ Ratio	0.89 + 0.02	1.04 + 0.04*
Peak Ca2+ Ratio	0.50 + 0.05	0.42 + 0.07
Ca2+ Transient Rate (D/sec)	45.24 + 5.85	40.53 + 10.95
Ca2+ Decay Rate (D/s)	-4.91 + 0.99	-2.87 + 0.57
Time to peak Ca2+ (ms; TTP)	34 + 2	41 + 3
Time to 50% Ca2+ Decay(ms; TTD50%)	101 + 8	117 + 6

Table 2: Contractile function and Ca²⁺ transients in adult rat myocytes 4 days after cTnIT144D gene transfer. An analysis of contractile function (upper panel) and Ca²⁺ transients (lower panel) is shown in myocytes after gene transfer of cTnIT144D compared to wild-type cTnI. Myocytes are isolated from 2-3-month-old adult rats, and data are presented as mean ± SEM (n = number of myocytes). Statistical comparisons of contractile function (left) and Ca²⁺ transients (right) are performed using an unpaired Student's t-test with significance set to *p < 0.05.

Supplemental Figure 1: Components of the pacing system for myocytes plated on laminin-coated CSs. (A) Custom pacing chamber containing platinum electrodes in each chamber. (B) Pacing chamber with the first four chambers filled with media. (C) Pacing chamber attached to banana-jack cables, which are connected to the chamber and (D) the stimulator. (E) The connection between the stimulator (right) and the 37 °C incubator (left). Please click here to download this File.

Supplemental Figure 2: Components needed for myocyte contractile function and/or Ca²⁺ transient measurements. (A) The contractile function platform showing each component, with the numbered items explained in more detail in the Table of Materials. Components in the platform include an anti-vibration table (#1), inverted brightfield microscope (#2,3), CCD camera (#4), CCD controller and xenon power supply (#5), dual emission light source (#6), temperature controller (#7), peristaltic pump (#8), insulated tube holder (#9), coverslip-mounted perfusion chamber (#10), and vacuum system (#11). (B) Additional components include the fluorescence interface (#12), chamber stimulator (#13), and PC computer (#14), which are explained in more detail in the Table of Materials. Close-up views of numbered items in panel A are shown in C–F. (C) View of the xenon power supply (left) and CCD controller (right). (D) Temperature controller. (E) Peristaltic pump. (F) View of the base for the CS perfusion chamber (black arrow), the platinum electrode mount (gray arrow), and the top mount (white arrow) with #0 pan-head screws. Please click here to download this File.

Discussion

The chronic pacing protocol outlined in step 1 extends the useful time for studying isolated myocytes and assessing the impact of longer treatments. In our lab, consistent results were obtained up to 4 days post isolation when measuring contractile function using sarcomere length on chronically paced myocytes. However, myocyte contractile function deteriorates quickly when using media older than 1 week to pace myocytes.

For contractile function studies, the data are collected at 37 °C, which is near the body temperature for rats. To optimize myocyte viability and obtain consistent traces for each myocyte, these experiments are performed at a pacing frequency lower than the physiological heart rate of ~5 Hz in rats. These settings produce consistent contractile function data, but chamber temperature and/or pacing frequency can be adjusted if the shortening traces remain consistent across multiple CSs containing the same treatment group of myocytes. In addition, optimal results are obtained by choosing myocytes that lack blebs, are fully adhered to a CS, and contract at both ends of a rod-shaped cell. Cells attached at only one end, those with multiple blebs, and/or myocytes contracting at only one end are not desirable and are often more difficult to record due to background movement. For these studies, ≥20 sarcomeres are included in the ROI to achieve a sharp power spectrum peak, reproducible resting sarcomere lengths, and an optimal signal/noise ratio for the shortening trace. A ROI with as few as seven sarcomeres can be used if the power spectrum peak remains sharp. The resting sarcomere length in isolated rat myocytes usually ranges from 2.00-1.80 μm. If a resting sarcomere length is less than 1.5 μm, an active contraction is not realistic based on our understanding of sarcomere architecture¹⁸ and usually is the result of sub-optimal myocyte orientation.

The measurement of contractile function with or without Ca²⁺ transient measurements in intact myocytes is a higher throughput approach requiring less investment in developing the technical expertise needed for force measurements in intact single cells¹¹. Intact myocyte studies also focus exclusively on myocyte function, without the contribution of other cell types in multi-cellular preparations². An emerging area in this field is the development of higher throughput analysis by simultaneously measuring shortening and/or Ca²⁺ transients in multiple intact myocytes to accelerate the screening of therapeutic agents at the cellular level prior to more extensive in vivo analysis.

Contractile function and Ca²⁺ transient measurements also are possible in intact myocytes isolated from other rodent models such as mice, although there are some important considerations and differences compared to rat myocytes. Mouse myocytes are viable in culture for 24-36 h, but viability progressively decreases in myocytes cultured for more than 48 h¹⁹. This truncated culture interval should be considered when using vector-based gene transfer, especially for myofilament proteins that have a half-life in days rather than hours²⁰. Unlike rat myocytes, the successful isolation and culture of mouse myocytes also depend on the addition of myosin ATPase inhibitors, such as 2,3-butanedione monoxime or blebbistatin, to culture media¹⁹. As a result, chronic pacing is not a viable option in mouse myocyte cultures. A longer equilibration time of 15-20 min is also needed to remove the functional impact of these myosin inhibitors on isolated mouse myocytes. Finally, mouse myocytes are optimally paced at 0.5 Hz to obtain reproducible shortening traces compared to the 0.2 Hz utilized for rat myocytes.

In studies on PO-treated rats, contractile dysfunction is consistently detected with low-frequency pacing in rat myocytes. A greater frequency-dependent reduction in shortening also is detected in myocytes after PO compared to sham rats when there is in vivo contractile dysfunction^5,13. During a study using a range of frequencies, doubling the pump speed to provide adequate media produced steady-state shortening within 15-20 s after changing the stimulation frequency between 0.2 and 2 Hz. Rat myocytes also respond to short-term stimulation at higher frequencies up to 8 Hz, although cell viability quickly deteriorates at these higher frequencies. Overall, myocyte contractile function has proven to be a reproducible approach to confirm or validate in vivo cardiac dysfunction in rat models, such as PO- compared to sham-treated rats (Table 1; see Kim et al.¹³). In addition, this platform can test whether a treatment targeting myocytes restores or impairs contractile function prior to pursuing more expensive and/or laborious in vivo approaches. Both acute delivery and longer exposure to therapeutic agents and/or after gene delivery during primary culture are possible using this approach. For example, gene transfer experiments to replace endogenous cTnI with cTnIT144D indicate no significant change in shortening amplitude in myocytes from sham rats. In contrast, cTnIT144D restores peak shortening amplitude toward sham values in myocytes from PO-treated rats (Table 1). An inherent weakness of this approach is the absence of the typical load present in myocytes under in vivo conditions. As a result, responses may differ from load-dependent functional responses, and the example experiment indicates that in vivo approaches are needed to further investigate whether cTnIT144D improves cardiac performance during PO.

The measurement of both shortening and Ca²⁺ transients in the same myocyte is an advantage of this platform. For example, the direction of change may differ for Ca²⁺ transients compared to contractile function. As shown in Table 2, peak shortening increases significantly after gene transfer of cTnIT144D into myocytes from 2-3-month-old rats. This outcome differs from the data obtained in older, sham-treated myocytes (Table 1) and may be related to rat age⁵. However, further testing is needed to prove this interpretation. The data in Table 2 also show that cTnIT144D does not change the peak Ca²⁺ amplitude and, instead, tends to slow the Ca²⁺ decay rate and elevate resting Ca²⁺. These findings suggest that cTnIT144D expression enhances contractile function, while the Ca²⁺ cycling machinery compensates to dampen this effect. These results highlight the ability of this approach to distinguish between changes in contractile function and Ca²⁺ cycling. While the specific results presented here are not yet definitive, they provide a solid rationale for developing a genetic animal model to express myocardial cTnIT144D and evaluate whether its expression blunts dysfunction caused by PO in the future. A final observation from the Ca²⁺ transient data is that fluorescent dyes such as Fura-2AM change the contractile function kinetics in myocytes²¹. Although the relative response produced by a given treatment or animal model is comparable within Fura-2-loaded myocytes, these results should not be combined with shortening measurements made in the absence of Fura-2AM. To optimize the use of Fura-2AM for Ca²⁺ transient analysis, the laboratory most often measures shortening in the absence of Fura-2AM and performs a subset of experiments to measure Ca²⁺ transients.

Materials

MEDIA
Bovine serum albumin	Sigma (Roche)	3117057001	Final concentration = 0.2% (w/v)
Glutathione	Sigma	G-6529	Final concentration = 10 mM
HEPES	Sigma	H-7006	Final concentration = 15 mM
M199	Sigma	M-2520	1 bottle makes 1 L; pH 7.45
NaHCO3	Sigma	S-8875	Final concentration = 4 mM
Penicillin/streptomycin	Fisher	15140122	Final concentration = 100 U/mL penicillin, 100 μg/mL streptomycin
REAGENTS SPECIFICALLY FOR Ca2+ IMAGING
Dimethylsulfoxide (DMSO)	Sigma	D2650
Fura-2AM	Invitrogen (Molecular Probes)	F1221	50 μg/vial;  Prepare stock solution of 1 mM Fura-2AM + 0.5 M probenicid in DMSO;  Final Fura2-AM concentration in media is  5 μM
Probenicid	Invitrogen (Fisher)	P36400	Add 7.2 mg probenicid (0.5 M) to 1 mM Fura-2AM stock; Final concentration in media is 2.5 mM
MATERIALS FOR RAT MYOCYTE PACING
#1 22  mm2 glass coverslips	Corning	2845-22
3 x 36 inch cables with banana jacks	Pomona Electronics	B-36-2	Supplemental Figure 1, panel C
37oC Incubator  with 95% O2:5% CO2	Forma	3110	Supplemental Figure 1, panel E.  Multiple models are appropriate
Class II A/B3 Biosafety cabinet with UV lamp	Forma	1286	Multiple models are appropriate
Forceps – Dumont #5 5/45	Fine Science Tools	11251-35
Hot bead sterilizer	Fine Science Tools	1800-45
Low magnification inverted microscope	Leica	DM-IL	Position this microscope adjacent to the incubator to monitor paced myocytes for contraction at the start of pacing and after media changes;  4X and 10X objectives recommended
Pacing chamber	Custom		Supplemental Figure 1, panel A. The Ionoptix C-pace system is a commercially available alternative or see 22
Stimulator	Ionoptix	Myopacer	Supplemental Figure 1, panel D.
MATERIALS FOR CONTRACTILE FUNCTION and/or Ca2+ IMAGING ANALYSIS			ID in Supplemental Figure 2 & Alternatives/Recommended Options
Additional components for Ca2+ imaging analysis	Ionoptix	Essential system components: — Photon counting system            –  Xenon power supply with dual excitation light source            –  Fluorescence interface	- The photon counting system contains a photomultiplier (PMT) tube and dichroic mirrorand is installed adjacent to the CCD camera  (panel A #4).                                                                      – The power supply for the xenon bulb light source (see panel A #5 and panel C, left) is integrated with a dual excitation interface (340/380 nm  excitation and 510 nm emission) shown in panel A #6.                                                                                                                                 – The fluorescence interface between the computer and  light source is shown in panel B, #12.
CCD camera with image acquisition  hardware and software (240 frames/s)	Ionoptix	Myocam with CCD controller	Myocam and CCD controller are shown in Supplemental Fig. 2, panel A #4  and  panel A #5 & panel C #5 (right), respectively.  The controller is integrated with a PC computer system (panel B #14).
Chamber stimulator	Ionoptix	Myopacer	Panel B, #13; Alternative:  Grass model S48
Coverslip mounted perfusion chamber	Custom chamber for 22 mm2 coverslip with silicone adapter and 2-4 Phillips pan-head #0 screws  (arrow, panel F)		Panel A #10 & panel F;   Chamber temperature is calibrated to 37oC using a TH-10Km probe and the TC2BIP temperature controller (see temperature controller).  Commercial alternatives:  Ionoptix FHD or C-stim cell  chambers; Cell MicroControls culture stimulation system
Dedicated computer & software for data collection and analysis of function/Ca2+ transients	Ionoptix	PC with Ionwizard PC board and software	Panel B, #14; Contractile function is measured using either SarcLen (sarcomere length) or SoftEdge (myocyte length) acquisition modules of the IonWizard software. The Ionwizard software also includes PMT acquisition software for ratiometric  Ca2+ imaging in Fura-2AM-loaded myoyctes. – A 4 post electronic rack mount cabinet and shelves are recommended for housing the somputer and cell stimulator.  The fluorescence interface for Ca2+ imaging also is housed in this cabinet (see below).
Forceps – Dumont #5 TI	Fine Science Tools	11252-40	Panel F
Insulated tube holder for media	Custom		Panel A #9; This holder is easily assembled using styrofoam & a pre-heated gel pack to keep media warm
Inverted brightfield microscope	Nikon	TE-2000S	Install a rotating turret for epi-fluorescence (Panel A #2) for Ca2+ imaging.   A deep red (590 nm) condenser filter also is recommended to minimize fluorescence bleaching during Ca2+ imaging.
Isolator Table	TMC Vibration Control	30 x 36 inches	Panel A, #1; Desirable:  elevated shelving, Faraday shielding
Microscope eyepieces & objective	Nikon	10X CFI eyepieces 40X water CFI Plan Fluor objective	Panel A #3; 40X objective:  n.a. 0.08; w.d. 2 mm.  A Cell MicroControls HLS-1 objective heater is mounted around the objective (see temperature controller below).          NOTE:  water immersion dispensers also are now available for water-based objectives.
Peristaltic pump	Gilson	Minipuls 3	Panel A #8 and panel E
small weigh boat	Fisher	08-732-112
Temperature controller	Cell MicroControls	TC2BIP	Panel A #7; Panel D. This temperature controller heats the coverslip chamber to 37oC.    A preheater and objective heater are recommended for this platform.  A Cell MicroControls HPRE2 preheater and  HLS-1 objective heater are controlled  by the TC2BIP temperature controller for our studies.
Under cabinet LED light with motion sensor	Sylvania	#72423 LED light	Recommended for data collection during Ca2+ transient imaging under minimal room light..  Alternative:  A clip on flashlight/book light with flexible neck – multiple suppliers are available.
Vacuum line with in-line Ehrlenmeyer flask & protective filter	Fisher	Tygon tubing – E363;  polypropylene Ehrlenmeyer flask – 10-182-50B; Vacuum filter – 09-703-90	Panel A #11