All hiPSC-cardiomyocytes were derived from the Gibco® Human Episomal iPSC
Line (Life Technologies, Carlsbad, CA), following a previously reported
protocol.4 (link) HiPSCs were maintained in
chemically defined conditions on plates coated with truncated human vitronectin (VTN-N) in
Essential 8™ medium (Life Technologies). For differentiation, hiPSCs
were plated as colonies at a low density on truncated VTN-N-coated 6-well plates in
Essential 8™ Medium. HiPSCs were grown to 80% confluency at
which point chemically defined directed differentiation was performed in RPMI 1640 medium
supplemented with 213 μg/mL L-Ascorbic Acid (Sigma-Aldrich, St. Louis, MO) and 500
μg/mL human serum albumin (ScienCell, Carlsbad, CA) (CDM3)4 (link) with the sequential application of CHIR 99021 (6
μM, d0–d1) followed by inhibitor of Wnt protein 2 (IWP2, 5 μM,
d3–d5; Thermo Fisher Scientific, Waltham, MA, USA). After two to three weeks of
differentiation, hiPSC-CMs were harvested using 0.25% Trypsin (Life Technologies)
and dispersed into single cells via trituration. HiPSC-cardiomyocytes were replated on
Matrigel-coated, #1.5 glass coverslips at approximately 26,000
cells/cm2. Aged hiPSC-cardiomyocytes were cultured for 12 months before being
trypsinized and replated. Aged cells were cultured for 6 days before staining and imaging
to correspond with replating time of PE-treated cells.
Neonatal rat ventricular myocytes (NRVMs) were a generous gift from Dr. Ulrike
Mende (Cardiovascular Research Center, Rhode Island Hospital) and were harvested in
accordance with all institutional and national guidelines for the care and use of
laboratory animals as approved by the Institutional Animal Care and Use Committee. NRVMs
and hiPSC-cardiomyocytes were given 72 hours to adhere and recover after replating and
before beginning stimulation by phenylephrine (PE). Cells received either PE (2
μM) with Timolol (to ensure α-adrenergic stimulation, 0.2
μM)32 (link) or media alone for 72
hours. For cytosolic volume experiments, hiPSC-cardiomyocytes were incubated with Alexa
Fluor® conjugated wheat germ agglutinin (WGA, 10 μg/mL, Thermo Fisher
Scientific) for 10 minutes prior to fixation. All cells were then rinsed with KCl (100 mM)
to induce diastole and fixed in 4% paraformaldehyde for 10 minutes at 4°C.
Cells were stored in DPBS at 4°C until stained.
Line (Life Technologies, Carlsbad, CA), following a previously reported
protocol.4 (link) HiPSCs were maintained in
chemically defined conditions on plates coated with truncated human vitronectin (VTN-N) in
Essential 8™ medium (Life Technologies). For differentiation, hiPSCs
were plated as colonies at a low density on truncated VTN-N-coated 6-well plates in
Essential 8™ Medium. HiPSCs were grown to 80% confluency at
which point chemically defined directed differentiation was performed in RPMI 1640 medium
supplemented with 213 μg/mL L-Ascorbic Acid (Sigma-Aldrich, St. Louis, MO) and 500
μg/mL human serum albumin (ScienCell, Carlsbad, CA) (CDM3)4 (link) with the sequential application of CHIR 99021 (6
μM, d0–d1) followed by inhibitor of Wnt protein 2 (IWP2, 5 μM,
d3–d5; Thermo Fisher Scientific, Waltham, MA, USA). After two to three weeks of
differentiation, hiPSC-CMs were harvested using 0.25% Trypsin (Life Technologies)
and dispersed into single cells via trituration. HiPSC-cardiomyocytes were replated on
Matrigel-coated, #1.5 glass coverslips at approximately 26,000
cells/cm2. Aged hiPSC-cardiomyocytes were cultured for 12 months before being
trypsinized and replated. Aged cells were cultured for 6 days before staining and imaging
to correspond with replating time of PE-treated cells.
Neonatal rat ventricular myocytes (NRVMs) were a generous gift from Dr. Ulrike
Mende (Cardiovascular Research Center, Rhode Island Hospital) and were harvested in
accordance with all institutional and national guidelines for the care and use of
laboratory animals as approved by the Institutional Animal Care and Use Committee. NRVMs
and hiPSC-cardiomyocytes were given 72 hours to adhere and recover after replating and
before beginning stimulation by phenylephrine (PE). Cells received either PE (2
μM) with Timolol (to ensure α-adrenergic stimulation, 0.2
μM)32 (link) or media alone for 72
hours. For cytosolic volume experiments, hiPSC-cardiomyocytes were incubated with Alexa
Fluor® conjugated wheat germ agglutinin (WGA, 10 μg/mL, Thermo Fisher
Scientific) for 10 minutes prior to fixation. All cells were then rinsed with KCl (100 mM)
to induce diastole and fixed in 4% paraformaldehyde for 10 minutes at 4°C.
Cells were stored in DPBS at 4°C until stained.
